Conjugated 4&#39;-desmethyl nucleoside analog compounds

ABSTRACT

Tetrahydrofuranyl compounds are provided that are functionalized to include pendant conjugate groups, and which are useful in diagnostic assays and as research reagents. Novel intermediates for the synthesis of the compounds are also provided.

This application is a continuation-in-part of U.S. Ser. No. 039,846,filed Mar. 30, 1993, now abandoned, which is a continuation-in-part ofU.S. Ser. No. 903,160, filed Jun. 24, 1992, now abandoned, and PCTApplication No. US 92/04294, filed May 1992, which both arecontinuations-in-part of U.S. Ser. No. 703,619, filed May 21, 1991, nowU.S. Pat. No. 5,378,825, issued Jan. 3, 1995, which is acontinuation-in-part of U.S. Ser. No. 566,836, filed Aug. 13, 1990, nowU.S. Pat. No. 5,223,618, issued Jun. 29, 1993 and of U.S. Ser. No.558,663, filed Jul. 27, 1990, now U.S. Pat. No. 5,138,045, issued Aug.11, 1992. U.S. Ser. No. 730,619 is also a continuation-in-part of PCTApplication No. US 91/05713, filed Aug. 12, 1991, which is acontinuation of U.S. Ser. No. 566,836, now U.S. Pat. No. 5,223,618issued Jun. 29, 1992.

FIELD OF THE INVENTION

This invention is directed to oligomeric compounds that arefunctionalized to include covalently bound groups. Specifically, theoligomeric compounds include a tetrahydrofuran moiety that isfunctionalized with a pendant conjugate group.

BACKGROUND OF THE INVENTION

Oligonucleotides and their analogs have been developed and used inmolecular biology in certain procedures as probes, primers, linkers,adapters, and gene fragments. Modifications to oligonucleotides used inthese procedures include labeling with non-isotopic labels, e.g.fluorescein, biotin, digoxigenin, alkaline phosphatase, or otherreporter molecules. Other modifications have been made to the ribosephosphate backbone to increase the nuclease stability of the resultinganalog. These modifications include use of methyl and other alkylphosphonates, phosphorothioates, phosphorodithioate, phosphoamidate andphosphotriester linkages, and 2'-O-methyl ribose sugar units. Furthermodifications include modification made to modulate uptake and cellulardistribution. Phosphorothioate oligonucleotides are presently being usedin human clinical trials for various disease states, including use asantiviral agents. In view of the success of these oligonucleotides forboth diagnostic and therapeutic uses, there exists an ongoing demand forimproved oligonucleotide analogs.

Oligonucleotides and like molecules can interact with native DNA and RNAin several ways. One of these is duplex formation between anoligonucleotide and a single stranded nucleic acid. A further method isvia triplex formation between an oligonucleotide and double stranded DNAto form a triplex structure.

Naturally occurring or synthetic oligonucleotides, together with hybridspecies having both synthetic and natural components, can collectivelybe referenced as "oligomeric compounds." Because of their properties,these oligomeric compounds are known to be useful in a number ofdifferent areas. They can be used as probes in cloning, blottingprocedures, and in applications such as fluorescence in situhybridization (FISH). Also, since local triplex formation inhibits genetranscription, such oligomeric compounds can be used to inhibit genetranscription. Labeled oligomers can also be used to directly map DNAmolecules, such as by tagging an oligomer with a fluorescent label andeffecting hybridization to complementary sequences in duplex DNA.Oligomers can also be used as identification tags in combinatorialchemical libraries as is disclosed in patent publication WO 94/08051 andOhlmeyer et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 10922-10926.

Considerable research is being directed to the application ofoligonucleotides and oligonucleotide analogs that bind complementary DNAand RNA strands for use as diagnostics, research reagents and potentialtherapeutics.

For most uses, it is desirable to append to oligomeric compounds groupsthat modulate or otherwise influence their activity or their membrane orcellular transport. One method of increasing such transport is by theattachment of a pendant lipophilic group. U.S. application Ser. No.117,363, filed Sep. 3, 1993, now pending entitled "Amine-DerivatizedNucleosides and Oligonucleosides", describes several alkylaminofunctionalities and their use in the attachment of such pendant groupsto oligonucleosides. Additionally, U.S. application Ser. No. 07/943,516,filed Sep. 11, 1992, now abandoned, "Novel Amines and Methods of Makingand Using the Same" and corresponding to published PCT application WO94/06815 describe other novel amine-containing compounds, and theirincorporation into oligonucleotides for, inter alia, the purposes ofenhancing cellular uptake, increasing lipophilicity, causing greatercellular retention and increasing the distribution of the compoundwithin the cell. U.S. application Ser. No. 08/116,801, filed Sep. 3,1993, now pending entitled "Thiol-Derivatized Nucleosides andOligonucleosides", describes nucleosides and oligonucleosidesderivatized to include a thioalkyl functionality, through which pendantgroups are attached.

Although each of the above-noted patent applications describe usefulcompounds, there remains a need in the art for additional stablecompounds that bind complementary DNA and RNA. There further remains aneed in the art for additional methods of attaching pendant groups tooligomeric compounds to further enhance or modulate their binding,cellular uptake, or other activity.

OBJECTS OF THE INVENTION

It is an object of the invention to provide novel nucleosides for use inattaching pendant conjugate groups to oligomeric compounds.

It is a further object of the invention to provide oligomeric compoundsthat have pendant intercalators, nucleic acid cleaving agents, cellsurface phospholipids, diagnostic agents, fluorescent agents and otherconjugate groups.

It is yet another object of the invention to provide improvements inresearch and diagnostic methods and materials for assaying bodily statesin animals, especially disease states.

It is a further object of the present invention to provide methods ofproducing these novel compounds.

These and other objects will become apparent from the followingdescription and accompanying claims.

SUMMARY OF THE INVENTION

The present invention provides compounds useful in diagnostic assays andas research reagents, as well as methods and intermediates for thepreparation thereof.

In one aspect of the invention there are provided compounds ofstructure: ##STR1## wherein: R_(L) is a group of formula:

    R.sub.C --[Y].sub.e --Z--

in which

Z is O, S or HN;

Y is a bivalent linker;

e is 0 or 1;

and

R_(C) is alkyl, alkenyl, alkynyl, O-alkyl, O-alkenyl, O-alkynyl, apolyamine, a polyether, asteroid molecule,

a reporter molecule, an aromatic lipophilic molecule,

a non-aromatic lipophilic molecule, a reporter enzyme, a peptide, aprotein, a water soluble vitamin, a lipid soluble vitamin, acarbohydrate, a terpene molecule,

a phospholipid, an intercalator, a cell receptor binding molecule, acrosslinking agent, or a porphyrin;

B_(x) is a nucleobase;

X is H, OH, O-alkyl, O-alkoxyalkyl, O-alkylamino or F;

Q is O, S, CH₂, CHF or CF₂ ; and

R_(D) is H, hydroxyl, an activated phosphorous group, a nucleoside, anactivated nucleotide, a nucleotide, an oligonucleotide, anoligonucleoside or a protected derivative thereof.

Compounds are also provided according to the invention having structure:##STR2## wherein: L is a group of formula:

    R.sub.7 --[W].sub.a --[S--(CH.sub.2).sub.q ].sub.u --[HN--(CH.sub.2).sub.n ].sub.t --[O--(CH.sub.2).sub.m ].sub.v --Z

in which

Z is O, S or HN;

t, u and v are each independently integers from 0 to 200;

W is the residue of a linking moiety, said linking moiety being selectedfrom the group consisting of acid chlorides, anhydrides, cyclicanhydrides, alkyl halides, organometallics, chloroformares, isocynates,hydrazines, acids, hydroxylamines, semicarbazides, thiosemicarbazides,hydrazones, hydrazides, trityl thiol, oximes, hydrazide-hydrazones,semicarbazones and semithiocarbazones;

a is 0 or 1;

m, n and q are each independently integers from 1 to 4; and

R₇ is R_(C), H or a protecting group; and

X, B_(X), R_(C) and R_(D) are defined as above.

In some preferred embodiments t and u are 0, or u and v are zero, or tand v are zero. In other preferred embodiments t and u are 0 and m is 2.In other preferred embodiments t and v are 0 and q is 2, or u and v are0 and n is 2. In even other preferred embodiments u is 0 and each of tand v are 1, and m and n are each 2.

Also provided according to the invention are compounds of structure:##STR3## wherein:

R_(C), B_(x), X, Q, and R_(D) are defined as above.

In the above structures, in some preferred compounds, R_(C) is apolyether or a polyamine. In other preferred embodiments, R_(C) is asteroid molecule, preferably cholic acid, deoxycholic acid,dehydrocholic acid, cortisone, digoxigenin, testosterone, cholesterol or3-trimethylaminomethylhydrazido cortisone.

In some preferred embodiments R_(C) is a water soluble vitamin,preferably thiamine, riboflavin, nicotinic acid, pyridoxal phosphate,pyridoxine, pyridoxamine, deoxypyridoxine, pantothenic acid, biotin,folic acid, 5'-deoxyadenosylcobalamin, inositol, choline or ascorbicacid.

In other preferred embodiments R_(C) is a lipid soluble vitamin,preferably a retinal, a retinol, retinoic acid, β-carotene, vitamin D,cholecalciferol, a tocopherol, or a phytol.

In some preferred embodiments R_(C) is a protein, preferably aphosphodiesterase, a peroxidase, a phosphatase or a nuclease. In otherpreferred embodiments R_(C) is a reporter molecule, preferably achromaphore, a fluorophore or a radiolabel-containing moiety. Preferredfluorophores include fluorescein, chrysine, anthracene and perylene.

In some preferred embodiments, X is H, OH, F, O-alkyl having from one tosix carbons, O-alkylamino having from one to six carbons orO-alkoxyalkyl having from one to six carbons. In certain more preferredembodiments X is H or OH mimicking natural deoxyribo and ribo sugaranalogs. In other more preferred embodiments X is F, O-alkyl having fromone to six carbons, alkylamino having from one to six carbons orO-alkoxyalkyl having from one to six carbons such that the compounds arehomologous to certain nucleotide species that have either greaternuclease resistance or high binding affinity.

In other preferred embodiments, R_(D) is H or OH; or R_(D) is anactivated phosphorous group, or R_(D) is an oligonucleotide.

The present invention also provides novel synthons useful for thepreparation of monomeric and polymeric conjugate compounds. In someembodiments these synthons have the structure: ##STR4## wherein J is aleaving group and Q, RD, B_(x) and X are as defined above. Preferably, Jis OH, SH, NH₂, trifluoromethylsulfonyl, methylsulfonyl, halogen,O-trichloroacetimidate, acyloxy, dialkyl phosphite,2,4,6-trichlorophenyl, p-toluenesulfonyl,4-dimethylaminoazobenzenesulfonyl or 5-dimethylaminonaphthalenesulfonyl.

Also provided are methods for forming a 5'-desmethyl conjugate oligomerhaving structure: ##STR5## Preferred methods comprise the steps of: (a)providing a first synthon having structure: ##STR6## and (b) contactingthe first synthon with a second synthon having structure: ##STR7## for atime and under reaction conditions sufficient to form the conjugatedoligomer; wherein R_(E) is an activated phosphorous group, W is H or ahydroxyl protecting group, k is an integer from 0 to 50, E is aphosphorous linking species including phosphodiester, phosphotriester,phosphorothioate, phosphorodithioate, alkyl phosphonates especiallymethyl phosphonates and phosphoramidates phosphorous linking groups, andQ, R_(L), B_(x) and X are as defined above.

The present invention also provides methods for forming a 5'-desmethylconjugated monomer having structure: ##STR8##

In certain embodiments these methods comprise the steps of providing asynthon having structure: ##STR9## and contacting this synthon with anactivated conjugating group. This contacting should be for a time andunder reaction conditions sufficient to form the conjugated oligomer.Preferably, J is OH, SH, NH₂, trifluoromethylsulfonyl, methylsulfonyl,halogen, O-trichloroacetimidate, acyloxy, dialkyl phosphite,2,4,6-trichlorophenyl, p-toluenesulfonyl,4-dimethylaminoazobenzenesulfonyl or 5-dimethylaminonaphthalenesulfonyl;more preferably trifluoromethylsulfonyl, methylsulfonyl, halogen oracyloxy.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention provides novel nucleosides for incorporation intonucleic acids. In one aspect, the invention provides nucleosides bearingpendant conjugate groups.

The term "nucleoside" refers to a unit composed of a heterocyclic baseand a sugar, generally a pentose sugar. In naturally occurringnucleosides, the heterocyclic base typically is guanine, adenine,cytosine, thymine or uracil, and the sugar is normally deoxyribose,i.e., erythro-pentofuranosyl, or ribose, i.e., ribopentofuranosyl.Synthetic sugars also are known, including arabino, xylo or lyxopentofuranosyl sugars and hexose sugars. Further synthetic sugar are the4'-deoxy-4'-thio sugars (both 2'-deoxy and ribo varieties) that havebeen described by Secrist et al., J. Med. Chem. 1991, 34, 2361-2366 andpatent application PCT/FR93/00115, respectively, (whereby Q in the abovestructures is S). Throughout this specification, reference to the sugarportion of a nucleoside or other nucleic acid species shall beunderstood to refer to naturally occurring deoxyribose and ribose sugarsand also to sugar species which can replace the sugar moieties ofnaturally occurring nucleic acids.

Reference to the nucleobase or heterocyclic base portion of a nucleosideor other nucleic acid species shall be understood to refer tonaturally-occurring nucleobases, modified derivatives thereof, or tosynthetic nucleobases. Synthetic nucleobases are those which can replaceone or more of the naturally occurring base moieties of wild typenucleic acids.

Reference to inter-sugar linkages shall be taken to include moietiesthat join the sugar portions of nucleosides or nucleotides.

The term "nucleotide" refers to a nucleoside having a phosphate groupesterified to one of its 2', 3' or 5' sugar hydroxyl groups. Thephosphate group normally is a monophosphate, diphosphate ortriphosphate.

The term "oligonucleotide" refers to a plurality of monophosphatenucleotide units that are typically formed from naturally occurringbases and pentofuranosyl sugars joined by native phosphodiester bonds ina specific sequence. A homo-oligonucleotide is formed from nucleotideunits having the same heterocyclic base, e.g. polyadenosine.

The term "oligonucleotide analog" has been used in various publishedpatent application specifications and other literature to refer tomolecular species that are similar to oligonucleotides, but that havenon-naturally occurring portions. This term has been used to identifyoligonucleotide-like molecules that have altered sugar moieties, alteredbase moieties or altered inter-sugar linkages. Thus, the termoligonucleotide analog has been used to denote structures having alteredinter-sugar linkages including phosphorothioate, phosphorodithioate,methyl phosphonate, phosphotriester or phosphoramidate inter-nucleosidelinkages in place of phosphodiester inter-nucleoside linkages; purineand pyrimidine heterocyclic bases other than guanine, adenine, cytosine,thymine or uracil and sugars having other than the β pentofuranosylconfiguration or sugars having substituent groups at their 2' positionor substitutions for one or more of the hydrogen atoms. The term"modified oligonucleotide" also has been used in the literature todenote such structures.

The term "oligonucleotide mimic" has also been used to refer tomacromolecular moieties that function similarly to or "mimic" thefunction of oligonucleotides but have non-naturally occurringinter-sugar linkages. Oligonucleotide mimics thus can have natural oraltered or non-naturally occurring sugar moieties and natural or alteredor non-naturally occurring base moieties in combination withnon-naturally occurring dephospho linkages. Certain dephospho linkageshave been reviewed by Uhlmann, E. and Peyman, A., "OligonucleotideAnalogs Containing Dephospho Internucleo-side Linkages" in Methods inMolecular Biology, Chapter 16, Oligonucleotide Synthetic Protocols, S.Agrawal, Ed., The Humana Press, Inc., Totowa, N.J., 1993.

The term "oligomers" is intended to encompass oligonucleotides,oligonucleotide analogs, oligonucleosides or oligonucleotide-mimickingmacromolecules. Thus, "oligomers" refers to nucleosides or nucleosideanalogs that are joined together via either natural phosphodiester bondsor via other linkages.

In certain embodiments the nucleoside compounds of the invention lackthe 5'-methylene group present in conventional pentofuranosylnucleosides. In certain preferred embodiments a heteroatom occupies theposition normally occupied by the missing 5'-methylene group. In onesense, these compounds can be considered as 4'-desmethyl pentofuranosylnucleosides. However, in accordance with IUPAC rules, the lack of a5'-methylene carbon on the "sugar portion" of the nucleosides of theinvention require their designation as tetrahydrofuranyl moieties. Thus,in naming these compounds according to IUPAC rules, as for example inthe identification of the structural positions of the compound,established hierarchical or priority nomenclature rules are followed.Accordingly, in embodiments wherein a heteroatom has replaced the5'-methylene group, that heteroatom and the pendant conjugate groupattached thereto takes priority over the heterocyclic base of thenucleoside. In such structures, the tetrahydrofuranyl ring is numberedcounterclockwise and the position occupied by the heteroatom (in whatwould be the 5' position of a conventional nucleoside) is identified asthe 2-position. However, in identifying certain of the protons in theNMR spectra, conventional pentofuranosyl nucleoside numbering has beenused (except where otherwise noted) for the tetrahydrofuranylnucleosides.

For the purpose of this specification and the claims appended hereto,when an oligomeric structure of the invention is being considered, theends of this structure are referenced in the same manner as forconventional oligonucleotides. Thus, they are identified either as a 3'end or a 5' end. In other instances where analogy to conventionalpentofuranosyl nucleosides is made, strict IUPAC naming rules aredeviated from and the numbering system of the conventionalpentofuranosyl nucleosides is maintained. In these instances it isconvenient to consider certain tetrahydrofuranyl compounds more as4'-desmethyl pentofuranosyl compounds and thus attachment is noted asbeing at the 4' position.

Compounds of the invention include 4'-desmethyl nucleoside monomersmodified to bear conjugate groups on their 4'-carbons (i.e.,4'-desmethyl conjugate monomers) or oligomers which contain suchmodified nucleosides (4'-desmethyl conjugate oligomers). Some oligomericembodiments have the structure: ##STR10## wherein a pendant conjugategroup R_(L) is attached to the 4'-position of a nucleoside of theinvention. In this structure the "E" group can be any of the commonlyuse oligonucleotide phosphorous linkage including phosphodiester,phosphotriester, phosphorothioate, phosphorodithioate, alkyl phosphonateand phosphoamidate linkages. The pendant conjugate group can be a groupof formula:

    R.sub.C --[W].sub.a --[S--(CH.sub.2).sub.n ].sub.t --[O--(CH.sub.2).sub.m ].sub.v --Z--

wherein Z is O, NH or S, preferably O or NH, t, u and v are eachindependently integers from 0 to 200; m, n and q are each independentlyintegers from 1 to 4; W is the residue of a linking moiety, said linkingmoiety being selected from the group consisting of acid chlorides,anhydrides, cyclic anhydrides, alkyl halides, organometallics,chloroformares, isocynates, hydrazines, acids, hydroxylamines,semicarbazides, thiosemicarbazides, hydrazones, hydrazides, tritylthiol, oximes, hydrazide-hydrazones, semicarbazones andsemithiocarbazones; a is 0 or 1; and R_(C) is alkyl, alkenyl, alkynyl,O-alkyl, O-alkenyl, O-alkynyl, a polyamine, a polyether, asteroidmolecule, a reporter molecule, an aromatic lipophilic molecule, anon-aromatic lipophilic molecule, a reporter enzyme, a peptide, aprotein, a water soluble vitamin, a lipid soluble vitamin, acarbohydrate, a terpene molecule, a phospholipid, an intercalator, acell receptor binding molecule, a crosslinking agent, an RNA cleavingcomplex, a metal chelator, an alkylator or a porphyrin. desmethylnucleoside unit incorporated therein has been derivatized to bear apendant conjugate group as described above. When so derivatized, theoligomeric compound is useful, for example, in a diagnostic or otherpreparation that includes a nucleic acid binding agent to assist inidentification of the oligomeric compound, to aid in transfer of thecompound across cellular membranes, or to impart other properties. Sucha diagnostic or nucleic acid binding agent is formed from an oligomericcompound of the invention wherein the oligomeric compound includesmonomeric units bearing natural or non-natural occurring bases in asequence that is complementary to and will specifically hybridize with aregion of an RNA or DNA of interest.

For the purpose of identification, a functionalized oligomeric compoundaccording to the invention can be characterized as a substituent-bearing(e.g., steroidbearing) oligomeric compound. Such oligomeric compoundswill have at least one pendant group attached thereto to modulate theiractivity.

The compounds of the inventions have various uses including being usefulas research reagents and in diagnostic assays. In one particulardiagnostic assay, compounds of the inventions are used to isolate theeffects of one cellular adhesion molecule from those of a furthercellular adhesion molecule in a protein expression assay. In this assay,the effect of induced expression of intercellular adhesion molecule-1(ICAM-1) were diminished while those of VCAM-1 were maintained. Thisallows for analysis of the VCAM-1 protein expression without concurrentexpression and interference of ICAM-1 protein.

In general, the oligomeric compounds bearing pendant conjugate groups ofthe present invention can be used to bind to various other targetmolecules. Target molecules of the present invention can include any ofa variety of biologically significant molecules. Other such targetmolecules can be nucleic acids, carbohydrates, glycoproteins or otherproteins.

In binding to nucleic acids, the functionalized oligomeric compounds ofthe invention bind with complementary strands of RNA or DNA. Afterbinding, the oligomeric compound and the RNA or DNA strand can beconsidered to be complementary strands which are "duplexed" in a manneranalogous to native, double-stranded DNA. In such complementary strands,the individual strands are positioned in such a manner with respect toone another so as to allow Watson-Crick type, Hoogsteen type or anothertype hybridization of the heterocyclic bases of one strand to theheterocyclic bases of the opposing strand.

Binding to nucleic acids can be practiced against nucleic acids from avariety of sources including organisms ranging from unicellularprokaryotes and eukaryotes to multicellular eukaryotes. The nucleic acidfrom any organism that utilizes DNA-RNA transcription or RNA-proteintranslation as a fundamental part of its hereditary, metabolic orcellular control is susceptible to such binding. Seemingly diverseorganisms such as bacteria, yeast, virus, protozoa, algae, all plant andall higher animal forms, including warm-blooded animals, are sources ofsuch nucleic acid. Further, since each of the cells of multicellulareukaryotes includes both DNA-RNA transcription and RNA-proteintranslation as an integral part of their cellular activity, nucleic acidbinding for various purposes, e.g. diagnostics, can also be practiced onsuch cellular populations. Furthermore, many of the organelles, e.g.,mitochondria and chloroplasts, of eukaryotic cells include transcriptionand translation mechanisms. Thus, single cells, cellular populations ororganelles can also be included within the definition of organisms thatare capable of being treated with nucleic acid binders. In somepreferred embodiments of the present invention, the target molecule is aprotein such as an immunoglobulin, receptor, receptor binding ligand,antigen or enzyme and more specifically can be a phospholipase, tumornecrosis factor, endotoxin, interleukin, plasminogen activator, proteinkinase, cell adhesion molecule, lipoxygenase, hydrolase or transacylase.In other embodiments of the invention the target molecules can beimportant regions of the human immunodeficiency virus, Candida, herpesviruses, papillomaviruses, cytomegalovirus, rhinoviruses, hepatitisviruses, or influenza viruses. In further embodiments, the targetmolecules can be regions of an oncogene. In still further embodiments,the target molecule is ras 47-mer stem loop RNA, the TAR element ofhuman immunodeficiency virus or the gag-pol stem loop of humanimmunodeficiency virus (HIV). Still other targets can induce cellularactivity. For example, a target can induce interferon.

Binding also can be practiced against transcription factors. In bindingto transcription factors or other target molecules, the transcriptionfactor or other target molecule need not be purified. It can be present,for example, in a whole cell, in a humoral fluid, in a crude celllysate, in serum or in other humoral or cellular extracts. Of course,purified transcription factor or a purified form of another targetmolecule is also useful in some aspects of the invention.

In still other embodiments of the present invention, syntheticallyprepared transcription factors or other target molecules can be useful.A transcription factor or other target molecule also can be modified,such as by biotinylation or radiolabeling. For example, a syntheticallyprepared transcription factor can incorporate one or more biotinmolecules during synthesis or can be modified post-synthesis.

Transcription factors, as the term is used herein, are DNA- orRNA-binding proteins that regulate the expression of genes. HIV tat andc-rel are examples of transcription factors which regulate theexpression of genes. Also encompassed by the term are DNA and RNAbinding proteins which are not strictly considered transcriptionfactors, but which are known to be involved in cell proliferation. Thesetranscription factors include c-myc, fos, and jun. Methods of thepresent invention are particularly suitable for use with transcriptionfactors as target molecules since transcription factors generally occurin very small cellular quantities.

In still other embodiments of the invention, nucleic acid binding can beused to treat objects (glasswares, petri dishes, instruments or thelike) to sterilize, sanitize or disinfect such objects that may harboran organism, e.g., bacterial, protozoan, viral, fungal or otherinfectious or non-infectious agent on the object. In binding to thenucleic acid of the agent, the compounds of the invention will kill,abrogate, inhibit, curtail or other wise control, eradicate or renderharmless the cellular growth or gene expression of such organisms on theobject. Such nucleic acid binding can also be used to render suchorganisms incapable or otherwise unable to reproduce themselves.

In still further embodiments of the inventions, the nucleic acid bindingproperties of the compounds of the invention can be used to form duplexstructures with an unknown oligomer species for identification of thatspecies by gel analysis including slab and capillary gelelectrophoresis. In still further embodiments of the inventions, thecompounds of the invention can be used as tags in identification ofother compounds in combinatorial libraries.

Preferred nucleobase units for oligomeric compounds of the inventioninclude naturally occurring or synthetic purine or pyrimidineheterocyclic bases, including but not limited to adenine, guanine,cytosine, thymine, uracil, 5-methylcytosine, hypoxanthine or2-aminoadenine. Other such heterocyclic bases include 2-methylpurine,2,6-diaminopurine, 6-mercaptopurine, 2,6-dimercaptopurine,2-amino-6-mercaptopurine, 5-methylcytosine,4-amino-2-mercaptopyrimidine, 2,4-dimercaptopyrimidine and5-fluorocytosine. Representative heterocyclic bases are disclosed inU.S. Pat. No. 3,687,808 (Merigan, et al.), which is incorporated hereinby reference.

In preferred embodiments, the oligomeric compounds of the inventioninclude from about 2 to about 50 nucleoside subunits (i.e., k=about 1 toabout 49).

As used in this specification, alkyl groups of the invention include butare not limited to C₁ -C₁₂ straight and branched chained alkyls such asmethyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl,decyl, undecyl, dodecyl, isopropyl, 2-butyl, isobutyl, 2-methylbutyl,isopentyl, 2-methylpentyl, 3-methylpentyl, 2-ethylhexyl and2-propylpentyl. Alkenyl groups include but are not limited tounsaturated moieties derived from the above alkyl groups including butnot limited to vinyl, allyl and crotyl. Alkynyl groups includeunsaturated moieties having at least one triple bond that are derivedfrom the above alkyl groups including but are not limited to ethynyl andpropargyl. Alkanoyl groups according to the invention are alkyl, alkenylor alkynyl groups attached through a carbonyl group.

The term aryl is intended to denote monocyclic and polycyclic aromaticgroups including, for example, phenyl, naphthyl, xylyl, pyrrole, andfuryl groups. Although aryl groups (e.g., imidazo groups) can include asfew as 3 carbon atoms, preferred aryl groups have 6 to about 14 carbonatoms, more preferably 6 to about 10 carbon atoms. Aralkyl and alkarylgroups according to the invention each include alkyl and aryl portions.Aralkyl groups are attached through their alkyl portions, and alkarylgroups are attached through their aryl portions. Benzyl groups provideone example of an aralkyl group, and p-tolyl provides an example of analkaryl group.

Alkylamino and aminoalkyt groups according to the invention each includeamino and alkyl portions. Alkylamino groups are attached through theiramino portions, and aminoalkyl groups are attached through their alkylportions. Methylamino groups provide one example of an alkylamino group,a β-aminobutyl group is one example of an aminoalkyl group.

The terms alkyl, alkaryl, aralkyl and aryl are intended to denote bothsubstituted (e.g., halogenated and hydroxylated) and unsubstitutedmoieties.

Halogens include fluorine, chlorine and bromine.

Suitable heterocyclic groups include but are not limited to imidazole,tetrazole, triazole, pyrrolidine, piperidine, piperazine and morpholine.Heterocycloalkyl groups are cyclic alkyl groups containing a heretoatom.Heterocycloalkaryl groups are aryl heterocycles bearing at least onealkyl substituent.

Amines include amines of all of the above alkyl, alkenyl and aryl groupsincluding primary and secondary amines and "masked amines" such asphthalimide. Amines are also meant to include polyalkylamino compoundsand aminoalkylamines such as aminopropylamine and furtherheterocycloalkylamines such as imidazol-1, 2 or 4-yl-propylamine.

Substituent groups for the above as well as for other moieties listedbelow include but are not limited to other alkyl, haloalkyl, alkenyl,alkynyl, alkoxy, thioalkoxy, haloalkoxy and aryl groups as well ashalogen, hydroxyl, amino, azido, carboxy, cyano, nitro, mercapto,sulfides, sulfones, sulfoxides, keto, carboxy, nitrates, nitrites,nitroso, nitrile, trifluoromethyl, O-alkyl, S-alkyl, NH-alkyl, amino,silyl, amides, ester, ethers, carbonates, carbamates, ureas, imidazoles,intercalators, conjugates, polyamines, polyamides, polyethylene glycols,polyethers, groups that enhance the pharmacodynamic properties ofoligonucleotides, and groups that enhance the pharmacokinetic propertiesof oligonucleotides. Other suitable substituent groups also includerhodamines, coumarins, acridones, pyrenes, stilbenes,oxazolopyridocarbazoles, anthraquinones, phenanthridines, phenazines,azidobenzenes, psoralens, porphyrins and cholesterols. One particularlypreferred group is CF₃.

As used in the present invention, the term polyether means a linear orbranched alkyl chain periodically interrupted by oxygen atoms. Onepreferred example of a polyethers is polyethylene glycol. The termpolyamine as used herein includes the nitrogen analogs of suchstructures, and the term polythioether includes the sulfur analogs ofsuch structures.

Carbohydrates according to the invention are inclusive of pentose,hexose and higher sugars, and the polymeric species thereof.Representative carbohydrates include glucose and galactose and theirderivatives including, as for example, glycals, glycal epoxides andglycosides.

Terpenes are known in the art as oligomers of isoprene, particularly thedipentenes, pinenes, and myrcenes. Included within the definition ofterpene molecules are terpene derivatives such as camphor and menthol.

The term phospholipid as used herein includes those compounds which uponhydrolysis yield phosphoric acid, an alcohol and one or more fattyacids. Representative examples of phospholipids include lecithin,cephalin and sphingomyelin.

As used in the present invention, groups that enhance thepharmacodynamic properties include groups that improve oligonucleotideuptake, enhance oligonucleotide resistance to degradation, and/orstrengthen sequence-specific hybridization with RNA. Groups that enhancethe pharmacokinetic properties, in the context of this invention,include groups that improve oligonucleotide uptake, distribution,metabolism or excretion.

For the purposes of this invention, the terms "reporter molecule" and"reporter enzyme" are inclusive of those molecules or enzymes that havephysical or chemical properties that allow them to be identified ingels, fluids, whole cellular systems, broken cellular systems and thelike utilizing physical properties such as spectroscopy, radioactivity,colorimetric assays, fluorescence, and specific binding. Particularlyuseful as reporter molecules are fluorophores, chromaphores andradiolabel-containing moieties. Fluorophores are molecules detectable byfluorescence spectroscopy. Examples of preferred fluorophores arefluorescein and rhodamine dyes and acridines. There are numerouscommercial available fluorophores including "Texas Red" and other likefluoresceins and rhodamines available for Molecular Probes, Eugene, OR.Chromaphores are molecules capable of detection by visible orultraviolet (UV-VIS) absorbance spectroscopy. Examples of chromaphoresare polynuclear aromatics such as anthracene, perylene, pyrene,rhodamine and chrysene. Radiolabel-containing moieties, as used herein,are molecules incorporating at least one radioactive atom, such as ³ Hor ¹⁴ C, enabling detection thereby. Reporter enzymes may be detecteddirectly or via their enzymatic products by any of the methods mentionedabove. Particularly useful as reporter enzymes are alkaline phosphataseand horseradish peroxidase.

Steroid molecules according to the invention include those chemicalcompounds that contain a perhydro-1,2-cyclopentanophenanthrene ringsystem. Particularly useful as steroid molecules are the bile acidsincluding cholic acid, deoxycholic acid and dehydrocholic acid; steroidsincluding cortisone, digoxigenin, testosterone and cholesterol andcationic steroids such as cortisone having a trimethylaminomethylhydrazide group attached via a double bond at the 3-position of thecortisone ring (3-trimethylaminomethylhydrazido cortisone).

Proteins and peptides are utilized in their usual sense as polymers ofamino acids. Normally peptides comprise such polymers that contain asmaller number of amino acids per unit molecule than do the proteins.Particularly useful as peptides and proteins are sequence-specificpeptides and proteins including phosphodiesterases, peroxidases,phosphatases and nucleases. Such peptides and proteins include, but arenot limited to, SV40 peptide, RNase A, RNase H and Staphylococcalnuclease.

Lipophilic molecules include naturally-occurring and synthetic aromaticand non-aromatic moieties such as fatty acids, esters and alcohols,other lipid molecules, cage structures such as adamantane andbuckminsterfullerenes, and aromatic hydrocarbons such as benzene,perylene, phenanthrene, anthracene, naphthalene, pyrene, chrysene, andnaphthacene. Particularly useful as lipophilic molecules are alicyclichydrocarbons, saturated and unsaturated fatty acids, waxes, terpenes andpolyalicyclic hydrocarbons including adamantane andbuckminsterfullerenes.

Alkylators according to the invention are moieties that can effectattachment of electrophilic groups to targeted molecular structures.Representative alkylators are disclosed by Meyer, et al., J. Am. Chem.Soc. 1989, 111, 8517.

Intercalators are polycyclic aromatic moieties that can insert betweenadjacent base pairs without affecting normal Watson-Crick base pairing,and include hybrid intercalator/ligands such as thephotonuclease/intercalator ligand6-[[[9-[[6-(4-nitrobenzamido)hexyl]amino]acridin-4-yl]carbonyl]amino]hexanoylpentafluorophenylester. This compound has two noteworthy features: an acridine moietythat is an intercalator and a p-nitrobenzamido group that is aphotonuclease. Other representative intercalators are disclosed byManoharan, M., Antisense Research and Applications, Crooke and Lebleu,eds., CRC Press, Boca Raton, 1993.

Cell receptor binding molecules according to the invention are vitaminsand carbohydrate moieties for which specific receptors exist within acell. Representative cell receptor binding molecules are disclosed byapplication Ser. No. PCT/US92/09196, filed Oct. 23, 1992, the contentsof which are incorporated herein by reference.

Crosslinking agents are moieties that can effect intrastrand orinterstrand covalent binding of RNA and/or DNA, and includephoto-crosslinking agents. Examples of photocrosslinking agents includearyl azides such as N-hydroxysuccinimidyl-4-azidobenzoate (HSAB) andN-succinimidyl-6(-4'-azido-2'-nitrophenylamino)hexanoate (SANPAH). Arylazides conjugated to oligomers will effect crosslinking with nucleicacids and proteins upon irradiation. Other representative crosslinkingagents are disclosed in International Patent Application Serial No.PCT/US93/02059, filed Mar. 5, 1993, which is incorporated herein byreference.

Useful crown amines are disclosed by Studer, et al., Helv. Chim. Acta1986, 69, 2081 and Smith-Jones, et al., Bioconjugate Chem. 1991, 2, 415.

Vitamins according to the invention generally can be classified as watersoluble or lipid soluble. Water soluble vitamins include thiamine,riboflavin, nicotinic acid or niacin, the vitamin B₆ pyridoxal group,pantothenic acid, biotin, folic acid, the B₁₂ cobalamin coenzymes,inositol, choline and ascorbic acid. Lipid soluble vitamins include thevitamin A family, vitamin D, the vitamin E tocopherol family and vitaminK (and phytols). The vitamin A family, including retinoic acid andretinol, are absorbed and transported to target tissues through theirinteraction with specific proteins such as cytosol retinol-bindingprotein type II (CRBP-II), Retinol-binding protein (RBP), and cellularretinol-binding protein (CRBP). These proteins, which have been found invarious parts of the human body, have molecular weights of approximately15 kD. They have specific interactions with compounds of the vitamin Afamily, especially, retinoic acid and retinol.

The vitamin A family of compounds can be attached to oligomers of theinvention via acid or alcohol functionalities found in the variousfamily members. For example, conjugation of an N-hydroxysuccinimideester of an acid moiety of retinoic acid to an amine function of apendant group of the oligomer results in linkage of the vitamin Acompound to the oligomer, via an amide bond. In similar fashion,standard esterification chemistries may be used to attach the acidmoiety of the retinoic acid group to a 4'-oxygen of a compound of theinvention, or to a hydroxyl function of a pendent group thereof.

α-Tocopherol (vitamin E) and other tocopherols (beta through zeta) canbe similarly conjugated to oligomers also to enhance uptake due to theirlipophilic character. The lipophilic vitamin, vitamin D, and itsergosterol precursors can be conjugated to oligomers through theirhydroxyl groups by first activating the hydroxyl groups by forming, forexample, hemisuccinate esters. Conjugation then is effected, as forinstance, to an aminolinker pendant from the oligomer, or through othersuitable functional groups described herein. Other vitamins that can beconjugated to oligomers through hydroxyl groups on the vitamins includethiamine, riboflavin, pyridoxine, pyridoxamine, pyridoxal,deoxypyridoxine. Lipid soluble vitamin K's and relatedquinone-containing compounds can be conjugated via carbonyl groups onthe quinone ring. The phytol moiety of vitamin K may also serve toenhance binding of the oligomers to cells.

Pyridoxal phosphate is the coenzyme form of Vitamin B₆. Vitamin B₆ isalso known as pyridoxine. Pyridoxal has specific B₆ -binding proteins.The role of these proteins in pyridoxal transport has been studied byZhang and McCormick, Proc. Natl. Acad. Sci. USA, 1991 88, 10407. Zhangand McCormick showed that a series of N-(4'-pyridoxyl)amines, in whichseveral synthetic amines were conjugated at the 4'-position ofpyridoxal, were able to enter cells by a process facilitated by the B₆transporter. Zhang and McCormick also demonstrated the release of thesesynthetic amines within the cell. Other pyridoxal family members includepyridoxine, pyridoxamine, pyridoxal phosphate, and pyridoxic acid.Pyridoxic acid, niacin, pantothenic acid, biotin, folic acid andascorbic acid can be conjugated to oligomers using N-hydroxysuccinimideesters as described above for retinoic acid.

Other groups for modifying properties of oligomers include RNA cleavingcomplexes, pyrenes, metal chelators, porphyrins, alkylators, hybridintercalator/ligands and photo-crosslinking agents. RNA cleavers includeo-phenanthroline/Cu complexes and Ru(bipyridine)₃ ²⁺ complexes. TheRu(bipyridine)₃ ²⁺ complexes interact with nucleic acids and cleavenucleic acids photochemically. Metal chelators include EDTA, DTPA, ando-phenanthroline. Alkylators include compounds such as iodoacetamide.Porphyrins include porphine, its substituted forms, and metal complexes.Pyrenes include pyrene and other pyrene-based carboxylic acids that canbe conjugated using protocols similar to those specified above.

Numerous suitable protecting groups are known in the art for protectingthe several functional groups of the compounds of the invention duringsynthesis. Protecting groups are known per se as chemical functionalgroups that can be selectively appended to and removed fromfunctionalities, such as hydroxyl groups and carboxyl groups. Thesegroups are present in a chemical compound to render such functionalityinert to chemical reaction conditions to which the compound is exposed.Protecting groups useful in the context of the present invention includebut are not limited to hydroxyl protecting groups such ast-butyldiphenylsilyl, t-butyldimethylsilyl, and dimethoxytrityl groups,thiol protecting groups such as S-trityl, S-p-methoxybenzylthioether,S-p-nitrobenzylthioether, and S-t-butylthioether. (see, e.g., Veber andHirschmann, et al., J. Org. Chem. 1977, 42, 3286 and Atherton, et al.,The Peptides, Gross and Meienhofer, Eds, Academic Press; New York, 1983;Vol. 9 pp. 1-38). Other representative protecting groups suitable forpractice in the invention may be found in Greene, T. W. and Wuts, P. G.M., "Protective Groups in Organic Synthesis" 2d. Ed., Wiley & Sons,1991.

In one aspect, the present invention is directed to nucleoside monomersthat bear at least one conjugate group at the 4'-position. In oligomericembodiments, the conjugate-bearing nucleoside is the 5'-terminal residueoligomer. In some preferred embodiments the conjugate group is apolyamine, and in other preferred embodiments the conjugate group is apolyether. In further preferred embodiments the conjugate group is apolythioether.

The conjugate groups of the preceding paragraph can further serve asbivalent linkers for other conjugate groups. Thus for instances, ahydroxyl, amino or thiol terminated polyether, polyamine orpolythioether is used a linker and the terminal group is then reactedwith a suitable functional group on a conjugate molecules to link theconjugate molecule to the polyether, polyamine or polythioether.Suitable functional groups for reacting with hydroxyl, amine or thiolgroups include a gamut of groups known in the organic chemical artsincluding, but not limited to, acid chlorides, anhydrides, cyclicanhydrides, alkyl halides, organometallics, chloroformates, isocynatesand the like. In a like manner other terminal groups can be utilized onthe linker including, but not limited to, hydrazines, aldehydes, acids,hydroxylamines, semicarbazides, thiosemicarbazides, hydrazones,hydrazides and the like. Protected forms of such compounds can also beused, as for example, protected aldehydes including, C-formyl,o-methylaminobenzenethio, aryl substituted imidazolidino moieties andthe like. In a like manner, protected thiol include trityl thiol thatcan be deblocked allowing a disulfide bonds to be formed between thelinker and the a sulfur bearing conjugate group to join the conjugategroup to the linker. Various nitrogen linkages can be used to join theconjugate groups including hydrazones, oximes, hydrazide-hydrazone,semicarbazone and semithiocarbazones.

The phrase polyamine species, including polyamines, as used hereinrefers to species that have a plurality of nitrogen atoms thereon.Polyamines include primary amines, hydrazines, semicarbazines,thiosemicarbazines and similar nitrogenous species. Such species can besymmetrical species such as polyamine containing polymers or they can beunsymmetrical wherein the amine functionalities of the polyamine areseparated in space by different moieties. In addition to carbon atomsother atomic species such as nitrogen and sulfur may also beincorporated into the polyamine species. In some preferred embodimentsof the invention, at least one nitrogen atom of the polyamine has a freeelectron pair.

Preferred as polyamine species are species that range in length fromabout 3 to about 20 units. More preferably species having at least onenitrogen atom and are of the general formula H₂ N[(CH₂)_(n) --(NH)_(y)]_(t) -- wherein n is an integer between 2 and 8 and t is an integerbetween 1 and 10. These species can be linear or cyclic. Cyclic amineswould include crown amines and mixed crown amines/crown ethers.

Other suitable nitrogen-containing compounds suitable for the formationof polyamine species include C₁ -C₂₀ straight chain alkylamine, C₁ -C₂₀straight chain substituted alkylamine, C₂ -C₅₀ branched chainalkylamine, C₂ -C₅₀ branched chain substituted alkylamine, C₃ -C₅₀cyclic alkylamine, C₃ -C₅₀ cyclic substituted alkylamine, C₂ -C₂₀straight chain alkenylamine, C₂ -C₂₀ straight chain substitutedalkenylamine, C₃ -C₅₀ branched chain alkenylamine, C₃ -C₅₀ branchedchain substituted alkenylamine, C₃ -C₅₀ cyclic alkenylamine, C₃ -C₅₀cyclic substituted alkenylamine, C₂ -C₂₀ straight chain alkynylamine, C₂-C₂₀ straight chain substituted alkynylamine, C₃ -C₅₀ branched chainalkynylamine, C₃ -C₅₀ branched chain substituted alkynylamine, C₃ -C₅₀cyclic alkynylamine, C₃ -C₅₀ cyclic substituted alkynylamine, C₁ -C₂₀straight chain alkylhydrazine, C₁ -C₅₀ straight chain substitutedalkylhydrazine, C₂ -C₅₀ branched chain alkylhydrazine, C₂ -C₅₀ branchedchain substituted alkylhydrazine, C₃ -C₅₀ cyclic hydrazoalkane, C₃ -C₅₀cyclic substituted hydrazoalkane, C₂ -C₂₀ straight chainalkenylhydrazine, C₂ -C₂₀ straight chain substituted alkenylhydrazine,C₃ -C₅₀ branched chain alkenylhydrazine, C₃ -C₅₀ branched chainsubstituted alkenylhydrazine, C₃ -C₅₀ cyclic hydrazoalkene, C₃ -C₅₀cyclic substituted hydrazoalkene, C₂ -C₂₀ straight chainalkynylhydrazine, C₂ -C₂₀ straight chain substituted alkynylhydrazine,C₃ -C₅₀ branched chain alkynylhydrazine, C₃ -C₅₀ branched chainsubstituted alkynylhydrazine, C₃ -C₅₀ cyclic hydrazoalkyne, C₃ -C₅₀cyclic substituted hydrazoalkyne, C₁ -C₂₀ straight chainalkylhydroxyamine, C₁ -C₂₀ straight chain substituted alkylhydroxyamine,C₂ -C₅₀ branched chain alkylhydroxyamine, C₂ -C₅₀ branched chainsubstituted alkylhydroxyamine, C₃ -C₅₀ cyclic oxyalkylamine, C₃ -C₅₀cyclic substituted oxyalkylamine, C₂ -C₂₀ straight chainalkenylhydroxyamine, C₂ -C₂₀ straight chain substitutedalkenylhydroxyamine, C₃ -C₅₀ branched chain alkenylhydroxyamine, C₃ -C₅₀branched chain substituted alkenylhydroxyamine, C₃ -C₅₀ cyclicoxyalkenylamine, C₃ -C₅₀ cyclic substituted oxyalkenylamine, C₂ -C₂₀straight chain alkynylhydroxyamine, C₂ -C₂₀ straight chain substitutedalkynylhydroxyamine, C₃ -C₅₀ branched chain alkynylhydroxyamine, C₃ -C₅₀branched chain substituted alkynylhydroxyamine, C₃ -C₅₀ cyclicoxyalkynylamine, C₃ -C₅₀ cyclic substituted oxyalkynylamine, C₁ -C₂₀straight chain alkylsemicarbazide, C₁ -C₂₀ straight chain substitutedalkylsemicarbazide, C₂ -C₅₀ branched chain alkylsemicarbazide, C₂ -C₅₀branched chain substituted alkylsemicarbazide, C₃ -C₅₀ cyclicalkylsemicarbazide, C₃ -C₅₀ cyclic substituted alkylsemicarbazide, C₂-C₂₀ straight chain alkenylsemicarbazide, C₂ -C₂₀ straight chainsubstituted alkenylsemicarbazide, C₃ -C₅₀ branched chainalkenylsemicarbazide, C₃ -C₅₀ branched chain substitutedalkenylsemicarbazide, C₃ -C₅₀ cyclic alkenylsemicarbazide, C₃ -C₅₀cyclic substituted alkenylsemicarbazide, C₂ -C₂₀ straight chainalkynylsemicarbazide, C₂ -C₂₀ straight chain substitutedalkynylsemicarbazide, C₃ -C₅₀ branched chain alkynylsemicarbazide, C₃-C₅₀ branched chain substituted alkynylsemicarbazide, C₃ -C₅₀ cyclicalkynylsemicarbazide, C₃ -C₅₀ cyclic substituted alkynylsemicarbazide,C₁ -C₂₀ straight chain alkylthiosemicarbazide, C₁ -C₂₀ straight chainsubstituted alkylthiosemicarbazide, C₂ -C₅₀ branched chainalkylthiosemicarbazide, C₂ -C₅₀ branched chain substitutedalkylthiosemicarbazide, C₃ -C₅₀ cyclic alkylthiosemicarbazide, C₃ -C₅₀cyclic substituted alkylthiosemicarbazide, C₂ -C₂₀ straight chainalkenylthiosemicarbazide, C₂ -C₂₀ straight chain substitutedalkenylthiosemicarbazide, C₃ -C₅₀ branched chainalkenylthiosemicarbazide, C₃ -C₅₀ branched chain substitutedalkenylthiosemicarbazide, C₃ -C₅₀ cyclic alkenylthiosemicarbazide, C₃-C₅₀ cyclic substituted alkenylthiosemicarbazide, C₂ -C₂₀ straight chainalkynylthiosemicarbazide, C₂ -C₂₀ straight chain substitutedalkynylthiosemicarbazide, C₃ -C₅₀ branched chainalkynylthiosemicarbazide, C₃ -C₅₀ branched chain substitutedalkynylthiosemicarbazide, C₃ -C₅₀ cyclic alkynylthiosemicarbazide, C₃-C₅₀ cyclic substituted alkynylthiosemicarbazide, C₁ -C₂₀ straight chainalkylhydrazone, C₁ -C₂₀ STRAIGHT chain substituted alkylhydrazone, C₂-C₅₀ branched chain alkylhydrazone, C₂ -C₅₀ branched chain substitutedalkylhydrazone, C₃ -C₅₀ cyclic hydrazoalkane, C₃ -C₅₀ cyclic substitutedhydrazoalkane, C₂ -C₂₀ straight chain alkenylhydrazone, C₂ -C₂₀ straightchain substituted alkenylhydrazone, C₃ -C₅₀ branched chainalkenylhydrazone, C₃ -C₅₀ branched chain substituted alkenylhydrazone,C₃ -C₅₀ cyclic hydrazoalkene, C₃ -C₅₀ cyclic substituted hydrazoalkene,C₂ -C₂₀ straight chain alkynylhydrazone, C₂ -C₂₀ straight chainsubstituted alkynylhydrazone, C₃ -C₅₀ branched chain alkynylhydrazone,C₃ -C₅₀ branched chain substituted alkynylhydrazone, C₃ -C₅₀ cyclichydrazoalkyne, C₃ -C₅₀ cyclic substituted hydrazoalkyne, C₁ -C₂₀straight chain alkylhydrazide, C₁ -C₂₀ straight chain substitutedalkylhydrazide, C₃ -C₅₀ branched chain alkylhydrazide, C₃ -C₅₀ branchedchain substituted alkylhydrazide, C₃ -C₅₀ cyclic alkylhydrazide, C₃ -C₅₀cyclic substituted alkylhydrazide, C₂ -C₂₀ straight chainalkenylhydrazide, C₂ -C₂₀ straight chain substituted alkenylhydrazide,C₃ -C₅₀ branched chain alkenylhydrazide, C₃ -C₅₀ branched chainsubstituted alkenylhydrazide, C₃ -C₅₀ cyclic alkenylhydrazide, C₃ -C₅₀cyclic substituted alkenylhydrazide, C₂ -C₂₀ straight chainalkynylhydrazide, C₂ -C₂₀ straight chain substituted alkynylhydrazide,C₃ -C₅₀ branched chain alkynylhydrazide, C₃ -C₅₀ branched chainsubstituted alkynylhydrazide, C₃ -C₅₀ cyclic alkynylhydrazide and C₃-C₅₀ cyclic substituted alkynylhydrazide.

As noted above, the conjugate groups of the invention may be attached tothe 4'-position of the nucleosidic sugar via a linker. Further linkinggroups include Ω-aminoalkoxy moieties and Ω-aminoalkylamino moieties forlinking a conjugate group to a 4'-hydroxyl group. Many other linkinggroups are known including many that are commercially available,including heterobifunctional and homobifunctional linking moietiesavailable from the Pierce Co. (Rockford, Ill.).

A further preferred group of linking groups include those groups used asthe internucleoside linkages between 4'-desmethyl nucleosides of theparent application, application Ser. No. 039,846, filed Mar. 30, 1993,now abandoned, the entire contents of which are herein expressedincorporated by reference.

In one embodiment for attaching a conjugate molecule to a 4-desmethylnucleoside, a linking moiety located on a 4-desmethyl nucleosidecompound of the invention may be reacted with an active ester derivativeof a conjugation moiety of the invention (e.g., cholic acid). Suchlinking moieties are particularly useful in extending polyalkylaminemoieties extending from the oligomeric compounds of the invention.Active ester derivatives are well known to those skilled in the art.Representative active esters include N-hydroxysuccinimide esters,tetrafluorophenolic esters, pentafluorophenolic esters andpentachlorophenolic esters. For cholic acid, the reaction of a pendantamino group and the active ester produces an oligomer in which cholicacid is attached to the N-terminal position through a linking group.Cholic acid or other such pendent groups can be attached to the carboxyterminal of the oligomeric compound by conversion to theN-hydroxysuccinimide ester thereof, and then by further reaction withthe oligomeric compound of interest.

Conjugate molecules of the invention can also be directly connected tothe 4'-desmethyl nucleosides compounds of the invention without use oflinkers. As for instance a conjugate molecule bearing a hydroxyl group,a thiol group, or an amino group is reacted with a 4'-desmethylnucleoside compound of the invention that has a leaving group at the 4'position. The OH, SH or NH₂ moiety of the conjugate groups displaces theleaving group to directly join the conjugate to the 4'-desmethylnucleoside compound of the invention.

The oligomeric compounds of the invention are synthesized utilizing atleast two different methods, both of which can use art recognized solidstate synthesis or solution phase synthesis. In a first process, the"4-desmethyl" nucleoside that is to be functionalized with a conjugategroup can be attached to a growing oligomeric compound and then sofunctionalized. In a second process the 4'-desmethyl nucleoside monomercan be first functionalized and then attached to the remainder of theoligomeric compound.

A conjugate group of a compound of the invention can be linked to a4'-desmethyl nucleoside monomer or oligomer through displacement of aleaving group from the 4'-position (as numbered using the sugar ringnumbering of a pentofuranosyl nucleoside) of a nucleoside, or the4'-terminal position (as numbered using the sugar ring numbering of apentofuranosyl nucleoside) of an oligonucleoside. In preferredembodiments, the 4'-desmethyl compound has structure: ##STR11## where Jis a leaving group. Leaving groups are functional moieties which aredesigned to be easily displaced. Representative leaving groups includealkyl and arylsulfonyls including p-toluenesulfonyl (tosyl),4-dimethylaminoazobenzenesulfonyl (dabsyl),5-dimethylaminonaphthalenesulfonyl (dansyl), trifluoromethylsulfonyl(triflate), methylsulfonyl (mesyl); halogens; o-trichloroacetimidates;2,4,6-trichlorophenyl; dialkyl phosphite and acyloxy leaving groupsincluding acetoxy, benzoyloxy, p-methoxybenzoyloxy andp-methylbenzoyloxy and other known leaving groups. Acyloxy leavinggroups (--OR_(E) where R_(E) is C(O)--) are preferred, particularlyOC(O)CH₃.

R_(D) can be H, hydroxyl, a nucleoside, an activated nucleotide, anucleotide, an oligonucleotide or an oligonucleoside or a protectedderivative thereof.

B_(X) is a naturally occurring or non-naturally occurring heterocyclicbase as described supra, and also includes such bases bearing protectinggroups.

The leaving group can be attached above or below the plane of the sugarring, i.e., cis or trans to the heretocyclic base. The leaving group maytherefore be displaced by either a bimolecular or unimolecularmechanism, dependent upon the specific leaving group and reactionconditions employed. In preferred embodiments the leaving group is transto the hetrocyclic base, and is displaced via a bimolecular nucleophilicdisplacement.

In preferred methods of the invention compounds having the above notedstructure can be incorporated into a wide variety of oligomericcompounds by standard chemistries. Thus, R_(D) can be an activatedphosphorous group. In the context of the invention activated phosphorousgroups are groups which are used to facilitate the creation ofinternucleosidic bonds in nucleic acid synthetic regimes. Thus,activated phosphorous groups include phosphate groups having P^(V) suchas, for example, activated phosphate esters, and phosphite groups havingP^(III) such as phosphoramidites or H-phosphonates. An activatednucleotide according to the invention is a nucleoside which bears anactivated phosphorous group.

In preferred processes of the invention a compound of the abovestructure is incorporated as the terminal nucleoside unit of a preformedoligomeric structure. Such oligomeric structures can be any of thevarious natural or synthetic oligonucleotides or oligonuleotide mimicsas known in the art.

In accordance with the present invention, methods which are amenable toautomated synthetic schemes, especially solid-state support syntheticschemes, are preferred. While a number of methodologies can be employed,one preferred methodology follows. A nucleoside analog is attached to asolid support in any conventional fashion. It is customary, andpreferred, to employ a linker to a solid support such as a polymericcarrier at the 3' position. An oligonucleotide or oligonucleoside isthen synthesized as per any of the various known solid statetechnologies. A final 4'-desmethyl nucleotide is then added to thegrowing oligomeric compound.

In one process of the invention, the 4'-desmethyl nucleotide issubstituted in the 4'-position with leaving group J as described above.The leaving group is then displaced with a linker or directly with aconjugate group of interest. If a linker is utilized the conjugate groupis then attached to the linker, either while still on the solid supportor post removal from the solid support.

Alternately the 4'-desmethyl nucleotide is first reacted with a linkeror directly with a conjugate group. The modified nucleotide is thenattached to the growing oligomeric compound on the solid support.

The 4'-desmethyl nucleoside (or nucleotide) is prepared with any base orbase analog, B_(x), and either a pentofuranosyl moiety, where Q isoxygen, a cyclopentane moiety or fluorinated analogue thereof where Q isCH₂, CHF or CF₂ or a 4'-deoxy-4'-thio moiety, where Q is S. In certainpreferred embodiments of the invention, the nucleoside is aribonucleoside having a 2'-hydroxyl functionality. Such a functionalitycan be any of various 2'-groups known in the art. If the 2'functionality is OH (a normal ribonucleotide), the 2'-hydroxyl groupscan be protected as necessary by means well known to persons of ordinaryskill in the art. The only requirement for this group and for itsprotection is that the same be designed so as to not interfere withsubstantive reactions in accordance with this invention. In otherpreferred embodiments, the 2'-hydroxyl group will be replaced with otherfunctional groups as for example 2'-alkoxy groups, 2'-alkoxy groups thatare substituted with other groups including imidazole and otherheterocyclic and amino groups, 2'-halogen, particularly fluoro.Particularly useful 2'-O-substituent groups include 2'-aminopropoxy,2'-imidazoylbutoxy, 2'-methoxy, 2'-ethoxy, 2'-propoxy, 2'-methoxyethoxyand 2'-deoxy-2'-fluoro.

Additional objects, advantages, and novel features of this inventionwill become apparent to those skilled in the art upon examination of thefollowing examples thereof, which are not intended to be limiting.

EXAMPLE 1 5'-O-tert-Butyldiphenylsilylthymidine, 2.

A stirring solution of thymidine (50.0 g, 207 mmol) and DMAP (10 mg,8.2×10⁻² mmol) in 400 mL of pyridine was treated with TBDPSCl (43.4 g,248 mmol) at 25° C. for 16 h. The solvent was removed under reducedpressure and the residue was diluted with 1 L of AcOEt. The mixture waswashed with 5% aqueous HCl (2×100 mL) and H₂ O (100 mL). The organiclayer was dried (MgSO₄) and concentrated under reduced pressure. Theproduct was purified by silica gel chromatography (CH₂ Cl₂ /MeOH 20:1)to give 87.3 g (88%) of 2 as a white solid. An analytical sample wascrystallized from diethylether. mp 164°-166° C. (170°-171° C. perMatulic-Adamic, J. Chem. Soc., Chem. Comm. 1985, 21, 1535) R_(f) (CH₂Cl₂ /MeOH 10:1) 0.31. ¹ H-NMR (CDCl₃): 1.08 (s, 9H, C--Me₃), 1.61 (s,3H, 5-Me), 2.18 (ddd, 1H, J=13.8, 8.5, 6.0 Hz, 2'H.sub.β), 2.19 (br s,1H, D₂ O exchangeable, 3'-OH), 2.44 (ddd, 1H, J=13.8, 5.6, 2.1 Hz,2'-H.sub.α), 3.25 (br s, 1H, 5'-OH, D₂ O exchangeable), 3.85 (dd, 1H,J=11.5, 2.5 Hz, 5'-CHH), 3.98 (dd, 1H J=11.5, 2.3 Hz, 5'-CHH), 4.06 (dd,1H, J=2.5, 2.3 Hz, 4'H), 4.55 (dd, 1H J=6.0, 5.6 Hz, 3'-H), 6.43 (dd,1H, J=8.5, 5.6 Hz, 1'-H), 7.26-7.51 (m, 6H, aromatic-H), 7.64-7.68 (m,5H, 6-H and aromatic-H), 9.57 (s, 1H, NH, D₂ O exchangeable).

EXAMPLE 2 N³ -Benzyloxymethyl-5'-O-tert-butyldiphenylsilylthymidine, 3.

To a stirred solution of 2 (117.0 g, 243.8 mmol) and Hunig's base(diisopropylethylamine, 63.0 g, 487.5 mmol) in CH₂ Cl₂ (400 mL) at 23°C. was added a solution of benzyl chloromethyl ether (40.7 g, 260.0mmol) over a 15 min. period. The resultant mixture was maintained at 23°C. and stirred for 14 h. Ether (1 L) was added to the mixture and theethereal solution was washed with 10% aqueous HCl (2×100 mL) and H₂ O(200 mL). The organic layer was dried (MgSO₄) and concentrated underreduced pressure. The residue was purified by silica gel columnchromatography (CH₂ Cl₂ /AcOEt 40:1 then 10:1) to yield 128.9 g (88%) of3 as a white solid. R_(f) (CH₂ Cl₂ /AcOEt 10:1) 0.31. ¹ H-NMR (CDCl₃):1.10 (s, 9H, C--Me₃), 1.65 (s, 3H, 5-Me), 2.16 (m, 1H, 2'-H.sub.β), 2.41(m, 1H, 2'-H.sub.β), 2.53 (br s, 1H 3'-OH), 3.84 (d, 1H, J=8.8 Hz,5'-CHH), 3.98 (d, 1H, J=8.8 Hz, 5'-CHH), 4.01 (s, 1H, 4'-H), 6.64 (br s,1H, 3'-H), 4.70 (s, 2H, OCH₂ Ph), 5.50 (s, 2H, NCH₂ O), 6.41 (dd, 1H,J=8.0, 5.8 Hz, 1'-H), 7.20-7.50 (m, 13H, aromatic-H), 7.65-7.69 (m, 3H,6-H and aromatic-H). ¹³ C-NMR (CDCl₃): 12.86 (-5-CH₃), 19.45 (+,C--Me₃), 27.09 (-, C--Me₃), 41.16 (+, 2'-C), 64.25 (+, 5'-C), 70.70 (+,O--C--Ph), 71.97 (-, 4'-C), 72.29 (+, N--C--O), 85.51 (-, 3'-C), 87.20(-, 1'-C), 110.47 (+, 5-C), 127.72, 128.08, 128.36 (-, aromatic-C),130.25 (-, 6-C), 132.42, 133.00 (+, aromatic-C) 134.39, 135.37, 135.62(-, aromatic-C), 137.95 (+, aromatic-C), 151.01 (+, 2-C), 163.68 (+,4-C).

EXAMPLE 3 N³-Benzyloxymethyl-3'-O-benzoyl-5'-O-tert-butyldiphenylsilyl-thymidine, 4.

A stirred solution of 3 (128.0 g, 213.3 mmol) in a 4:1 mixture of CH₂Cl₂ /Et₃ N (500 mL) was treated with (48.4 g, 40 mL, 344.6 mmol) of BzClat 23° C. for 8 h. The resultant precipitate was removed by filtration.The filtrate was concentrated under reduced pressure to leave the crudeproduct as a brownish syrup. Purification of the syrup by silica gelcolumn chromatography (hexanes/AcOEt 10:1 then 1:1) gave 130.7 g (87%)of 4 as a white solid. R_(f) (Hexanes/AcOEt 1:1) 0.82 ¹ H-NMR (CDCl₃):1.40 (s, 9H, C--Me₃), 1.60 (s, 3H, 5-Me), 2.37 (ddd, 1H, J=13.8, 9.3,7.2 Hz, 2'-H.sub.β), 2.62 (dd, 1H, J=13.8, 4.3 Hz, 2'-H.sub.β), 4.09 (m,2H, 5'-H), 4.26 (m, 1H, 4'-H), 4.74 (s, 2H, O--CH₂ --Ph), 5.54 (s, 2H,N--CH₂ --O), 5.71 (d, 1H, J=7.2 Hz, 3'-H), 6.57 (dd, 1H, J=9.3, 4.3 Hz,1'-H), 7.24-7.74 (m, 13H, aromatic-H), 8.05-8.15 (m, 3H, 6-H andaromatic-H). ¹³ C-NMR(CDCl₃): 12.82 (-, 5-Me), 19.54 (+, C--Me₃), 27.16(C--Me₃), 38.28 (+, 2'-C), 64.41 (+, 5'-C), 70.80 (+, O--C--Ph), 72.29(+, N--C--O), 75.60 (-, 4'-C), 85.28 (-, 1'-C and 3'-C), 110.95 (+,5-C), 127.72, 128.23, 128.37, 128.50, 128.63 (-, aromatic-C), 129.43 (+,aromatic-C), 129.84, 130.22 (-, aromatic-C), 132.14, 133.07 (+,aromatic-C), 133.60 (-, 6-C), 133.94, 135.32, 135.65 (-, aromatic-C),138.15 (+, aromatic-C), 151.19 (+, 2-C), 163.50 (+, 4-C), 166.11 (+,benzoyl C═O).

EXAMPLE 4 N³ -Benzyloxymethyl-3'-O-benzoylthymidine, 5.

The silyl ether 4 (96.0 g, 136.4 mmol) in THF (600 mL) was treated withhydrogen fluoride-pyridine (70% HF in pyridine, 30 mL) at 0° C. for 4 hunder a N₂ atmosphere. The resultant mixture was diluted with AcOEt (600mL) and washed with H₂ O (2×300 mL). The organic layer was dried (MgSO₄)and concentrated at reduced pressure. The residue was purified by silicagel column chromatography (CH₂ Cl₂ /AcOEt 10:1) to give 61.6 g (97%) of5 as a white solid. R_(f) (CH₂ Cl₂ /AcOEt 10:1) 0.29. ¹ H-NMR(CDCl₃ +D₂O): 1.95 (s, 3H, 5-Me), 2.53 (m, 2H, 2'-H), 4.00 (m, 2H, 5'-H), 4.25 (m,1H, 4'-H), 4.71 (s, 2H, O--CH₂ --Ph), 5.51 (s, 2H, N--CH₂ --O), 5.60 (m,1H 3'-H), 6.36 (dd, 1H, J=7.6, 6.6 Hz, 1'-H), 7.25-7.66 (m, 9H, 6 -H andaromatic-H), 8.05 (d, 2H, J=7.1 Hz, aromatic-H). ¹³ C-NMR(CDCl₃): 13.29(-, 5-Me), 37.82 (+, 2'-C), 62.54 (+, 5'-C), 70.73 (+, O--C--Ph), 72.25(+, N--C--O), 75.82 (-, 4'-C), 85.43 (-, 3'-C), 86.13 (-, 1'-C), 110.41(+, 5-C), 127.65, 128.36, 128.59 (-, aromatic-C), 129.34 (+,aromatic-C), 129.69 (-, 6-C), 133.60, 135.40 (-, aromatic-C), 137.87 (+,aromatic-C), 151.18 (+, 2-C), 163.65 (+, 4C), 166.11 (+, benzoyl C═O).

EXAMPLE 5 tert-Butyl N³-Benzoxymethyl-3'-O-benzoylthymidine-5'-carboxylate, 6

The reaction was performed as described by Corey and Samuelsson, J. Org.Chem. 1984, 49, 4735, for the oxidation of 5'-OH of the uridinederivative to its corresponding 5'-tert-butyl carboxylate. Chromium(VI)oxide (31.4 g, 314.2 mmol) in CH₂ Cl₂ (480 mL) was cooled to 0° C. andthen pyridine (49.7 g, 638.4 mmol) in DMF (120 mL) was added dropwise tothe reaction mixture (caution: extremely exothermic) over a period of 1h. The mixture was stirred at 0° C. for 30 min. Alcohol 5 (36.6 g, 78.5mmol) in CH₂ Cl₂ /DMF (4:1 v/v, 100 mL) was added followed by aceticanhydride (64.1 g, 628.4 mmol) and t-BuOH (116.4 g, 1.57 mmol). Theresultant mixture was warmed to 23° C. and stirred for 18 h. Ethanol (20mL) was added to the reaction and the mixture was stirred for additional15 min. The reaction mixture was poured into AcOEt (400 mL) and theinsoluble material was filtrated through a Buchner funnel padded with200 g of silica gel and 50 g of MgSO₄. The solid left in the funnel wasrinsed with AcOEt (4×100 mL). The combined filtrate and rinses wereconcentrated under reduced pressure. The residue was purified by silicagel column chromatography (hexanes/AcOEt 3:1) to give 25.6 g (61%) of 5as a white solid. An analytical sample (about 400 mg) was crystallizedfrom ether/hexanes afforded white, needle-like crystals. mp 80°-82° C.R_(f) (hexanes/AcOEt 3:1) 0.23. ¹ H-NMR(CDCl₃): 1.55 (s, 9H, C--Me₃),2.21 (ddd, 1H, J=14.3, 9.1, 5.0 Hz, 2'-H.sub.β), 2.61 (dd, 1H, J=14.3,5.2 Hz, 2'-H.sub.α), 4.63 (s, 1H, 4'-H), 4.71 (s, 2H, O--CH₂ --Ph), 5.51(s, 2H, N--CH₂ --O), 5.65 (d, 1H, J=5.0 Hz, 3'-H), 6.61 (dd, 1H, J=9.1,5.2 Hz, 1'-H), 7.24-7.63 (m, 8H, aromatic-H), 8.07 (d, 2H, J=7.1 Hz,aromatic-H), 8.09 (s, 1H, 6-H). ¹³ C--NMR(CDCl₃): 13.40 (-, 5-Me), 27.92(-, C--Me₃), 36.65 (+, 2'-C), 70.58 (+, O--C--Ph), 72.09 (+, N--C--O),76.66 (-, 4'C), 82.55 (-, 3'-C), 83.36 (+, C--Me₃), 86.88 (-, 1'-C),110.61 (+, 5-C), 127.55, 127.24 128.23, 128.53 (-, aromatic-C), 128.99(+, aromatic-C), 129.78 (-, 6-C), 133.71, 134.72 (-, aromatic-C), 138.06(+, aromatic-C), 151.14 (+, 2-C), 163.28 (+, 4-C), 165.26 (+, benzoylC═O), 169.40 (+, 5'-C).

EXAMPLE 6 N³ -Benzoxylmethyl-3'-O-benzoylthymidine-5'-carboxylic acid,7.

A solution of 6 (22.0 g, 41.0 mmol) in CF₃ COOH (100 mL) was stirred at23° C. for 2 h. Toluene (200 Ml) was then added and the mixture wasconcentrated under reduced pressure. The coevaporation of toluene wasrepeated twice to ensure complete removal of the CF₃ COOH. The resultantlight yellow powder was purified by silica gel column chromatography(CH₂ Cl₂ /AcOEt 8:1) to afford 19.3 g (87%) of 7 as a white powder.R_(f) (CHCl₃ /MeOH 4:1) 0.39, ¹ H-NMR (CDCl₃ +D₂ O): 1.95 (s, 3H, 5-Me),2.27 (ddd, 1H, J=14.3, 9.1, 4.9 Hz, 2'-H.sub.β), 2.68 (dd, 1H, J=14.3,5.2 Hz, 2'H.sub.α), 4.71 (s, 2H, O--CH₂ --Ph), 4.79 (s, 1H, 4'-H), 5.52(s, 2H, N--CH₂ --O), 5.76 (d, 1H, J=4.9 Hz, 3'-H), 6.55 (dd, 1H, J=9.1,5.2 Hz, 1'-H), 7.24-7.60 (m, 8H, aromatic-H), 7.97 (s, 1H, 6-H), 8.06(d, 2H, J=7.1 Hz, aromatic-H). ¹³ C-NMR(CDCl₃): 13.42 (+, 5-Me), 36.68(+, 2'-C), 70.83 (+, O--C--Ph), 73.38 (+, N--C--O), 76.93 (-, 4'-C),82.01 (-, 3'-C), 87.58 (-, 1'-C), 110.80 (+, 5-C), 127.72, 127.86,128.39, 128.70 (-, aromatic-C), 128.71 (+, aromatic-C), 129.90 (-, 6-C),133.95, 135.63 (-, aromatic-C), 128.53 (+, aromatic-C), 11.20 (+, 2-C),163.94 (+, 4-C), 165.61 (+, benzoyl C═O), 171.93 (+, 5'-C).

EXAMPLE 7(2'S,4'S,5'R)-1-[5'-Acetoxy-4'-benzoyloxy-tetrahydrofuran-2'-yl]-N³-benzoxymethylthymine, 8.

A stirred solution of 7 (10.6 g, 22.0 mmol) in DMF (75 mL) was treatedwith Pb (OAc)₄ (11.8 g, 26.5 mmol) at 23° C. for 2 h under darkness. Themixture was diluted with AcOEt (250 mL) and the resultant suspension wasfiltrated through a Celite pad (50 g). The solid was rinsed severaltimes with AcOEt. The combined filtrate and rinses were concentratedunder reduced pressure. The residue was purified by silica gel columnchromatography (hexanes/AcOEt 1:1) to give 6.5 g (60%) of a 3:7 α/β (asdetermined by the ¹ H-NMR 2'-H ratio) anomeric mixture of 8 as a lightyellow syrup. An aliquot of the anomeric mixture (˜0.2 g) was separatedon a silica gel column (hexanes/AcOEt 8:1→2:1 gradient) to afford 53 mgof 8α and 121 mg of 8β, both as white foams.

8α: R_(f) (hexanes/AcOEt, 1:1) 0.53. ¹ H-NMR (CDCl₃): 1.95 (d, 3H, J=1.1Hz, 5-CH₃), 2.05 (s, 3H, acetoxy-CH₃), 2.52-2.75 (m, 2H, 3'-H), 4.70 (s,2H, OCH₂ Ph), 5.49 (s, 2H, NCH₂ O), 5.60-5.65 (m, 1H, 4'-H), 6.40 (dd,1H, J=7.9, 3.8 Hz, 2'-H), 6.72 (d, 1H, J=4.0 Hz, 5'-H), 7.06 (d, 1H,J=1.1 Hz, 6-H), 7.22-7.60 (m, 8H, aromatic-H), 8.02 (d, 2H, J=7.6 Hz,aromatic-H). ¹³ C-NMR (CDCl₃): 13.04 (-, 5-CH₃), 20.73 (-, acetoxy-CH₃),33.81 (+, 3'-C), 70.45 (+, O--C--Ph), 71.08 (-,4'-C), 72.11 (+,N--C--O), 85.47 (-, 2'-C), 94.10 (-, 5'-C), 110.90 (+, 5-C), 127.47,128.11, 128.45 (-, aromatic-C), 128.64 (+, aromatic-C), 129.54 (-, 6-C),133.55, 133.92 (-, aromatic-C), 137.75 (+, aromatic-C), 150.55 (+, 2-C),163.01 (+, 4-C), 165.43 (+, benzoyl C═O), 169.18 (+, acetoxy C═O).

Anal. Calcd. for C₂₆ H₂₆ N₂ O₈ :C, 63.16; H, 5.26; N, 5.67. Found: C,63.12; H, 5.38; N, 5.45.

8β: R_(f) (hexanes/AcOEt 1:1) 0.47. ¹ H-NMR (CDCl₃): 1.95 (s, 3H,5-CH₃), 2.18 (s, 3H, acetoxy-CH₃), 2.33 (ddd, 1H, J=14.9, 8.2, 4.9 Hz,3'-H.sub.β), 2.75 (dd, 1H, J=14.9, 6.2 Hz, 3'-H.sub.α), 4.70 (s, 2H,OCH₂ Ph), 5.50 (s, 2H, NCH₂ O), 5.52 (d, 1H, J=4.9 Hz, 4'-H), 6.42 (s,1H, 5'-H), 6.73 (dd, 1H, J=8.2, 6.2 Hz, 2'-H), 7.22-7.63 (m, 9H, 6-H andaromatic-H), 8.04 (d, 2H, J=7.5 Hz, aromatic-H). ¹³ C-NMR (CDCl₃): 13.40(-, 5-CH₃), 20.82 (-, acetoxy-CH₃), 34.95 (+, 3'-C), 70.51 (+,O--C--Ph), 72.07 (+, N--C--O), 76.64 (-, 4'-C), 87.15 (-, 2'-C), 98.61(-, 5'-C), 111.03 (+, 5-C), 127.45, 128.11, 128.45 (-, aromatic-C),129.68 (-, 6-C), 133.02, 133.69 (-, aromatic-C). 137.77 (+, aromatic-C),150.91 (+, 2-C), 162.84 (+, 4-C), 165.12 (+, benzoyl C═O), 169.13 (+,acetoxy C═O). Anal. Calcd for C₂₆ H₂₆ N₂ O₈.H₂ O: C, 60.94; H, 5.47; N,5.47. Found: C, 60.98; H, 5.18; N, 5.30.

EXAMPLE 8(2'R,4'S,5'S)-(4'-Benzoxy-5'-O-polyethyleneglycoltetrahydrofuran-2'-yl)-N.sup.3-(benzyloxymethyl)thymine (9)

A solution of 3.7 g (7.5 mmol) of acetate 8, 25-35 mmol of PEG alcohol,and 4 mL (3.0 g, 30 mmol) of Et₃ N in 150 mL of CH₂ Cl₂ was added 14 mL(16.2 g, 75 mmol) of TMSOTf at -23° C. under Ar atmosphere. The reactionmixture was stirred at -23° C. for 2 h, then placed in the freezer (-15°C.) for 16-24 h. The resultant mixture was poured into a 500 mL bilayermixture of AcOEt/H₂ O (9:1) containing 15 mL of Et₃ N. The organic layerwas dried (MgSO₄) and concentrated at reduced pressure. The residue waspurified by SiO₂ column chromatography to yield 9.

9a: yield 84.9% (colorless syrup). R_(f) (hexanes/AcOEt 1:1) 0.22. ¹ HNMR (CDCl₃ +D₂ O) δ1.95 (s, 3H, 5-CH₃), 2.34 (ddd, J=14.3, 8.1, 5.1 Hz,3'-H.sub.α), 2.62 (dd, 1H, J=14.3, 6.5 Hz, 3'-H.sub.β), 3.60-3.95 (m,4H), 4.70 (s, 2H, OCH₂ Ph), 5.25 (s, 1H, 5'-H), 5.47 (d, J=5.1 Hz,4'-H), 5.50 (s, 2H, NCH₂ O), 6.80 (dd, 1H, J=8.1, 6.5 Hz, 2'-H),7.18-7.65 (m, 9H, 6-H and aromatic-H), 8.04-8.07 (m, 2H, aromatic-H). ¹³C NMR (CDCl₃ +D₂ O) δ13.16, 35.00 (3'-C), 61.41, 70.33, 70.76, 72.29,77.67 (4'-C), 86.53 (2'-C), 106.61 (5'-C), 111.56 (5-C), 127.66, 128.06,128.33 (6-C), 128.97, 129.84, 133.80, 134.38, 137.99, 151.41 (2-C),163.35 (4-C), 165.76 (benzoyl C═O).

9b: yield 84.3% (colorless syrup). R_(f) (hexanes/AcOEt 1:1) 0.18. ¹ HNMR (CDCl₃) δ1.95 (s, 3H, 5-CH₃), 2.34 (ddd, 1H, J=14.7, 8.1, 4.9 Hz,3'-H.sub.α), 2,62 (dd, 1H, J=14.7, 6.3 Hz, 3'-H.sub.δ), 3.36 (s, 3H, CH₃O), 3.54-3.98 (m, 12H), 4.72 (s, 2H, OCH₂ Ph), 5.24 (s, 1H, 5'-H), 5.49(d, 1H, J=4.9 Hz, 4'-H), 5.52 (s, 2H, NCH₂ O), 6.80 (dd, 1H, J=8.1, 6.3Hz, 2'-H), 7.26-7.65 (m, 9H, 6-H and aromatic-H), 8.02-8.06 (m, 2H,aromatic-H). ¹³ C NMR (CDCl₃) δ13.29, 35.06 (3'-C), 59.04 (CH₃ O),68.00, 70.14, 70.61, 71.91, 72.19, 77.51 (4'-C), 86.38 (2'-C), 106.43(5'-C), 111.43 (5-C), 127.65, 128.31, 128.60 (6-C), 129.07, 129.81,133.70, 134.45, 138.04, 151.41 (2-C), 163.28 (4-C), 165.51 (benzoylC═O).

9c: 76.5% yield (white foam). R_(f) (hexanes/AcOEt 1:1) 0.05. ¹ H NMR(CDCl₃) δ1.98 (s, 3H, 5-CH₃), 2.37 (ddd, 1H, J=14.7, 8.2, 4.9 Hz,3'-H.sub.α), 2,62 (dd, 1H, J=14.7, 6.5 Hz, 3'-H.sub.β), 2.78 (br s, 1H,OH, D₂ O exchangable), 3.57-3.99 (m, 16H), 4.72 (s, 2H, OCH₂ Ph), 5.25(s, 1H, 5'-H), 5.50 (d, 1H, J=4.9 Hz, 4'-H), 5.52 (s, 2H, NCH₂ O), 6.80(dd, 1H, J=8.2, 6.5 Hz, 2'-H), 7.26-7.65 (m, 9H, 6-H and aromatic-H),8.02-8.06 (m, 2H, aromatic-H). ¹³ C NMR (CDCl₃) δ13.27, 35.02 (3'-C),61.70, 68.02, 70.15, 70.28, 70.54, 70.73, 72.26, 72.61, 77.61 (4'-C),86.47 (2'-C), 106.55 (5'-C), 111.40 (5-C), 127.66, 128.31, 128.60 (6-C),129.12, 129.84, 133.71, 134.48, 138.04, 151.45 (2-C), 163.35 (4-C),165.61 (benzoyl C═O).

9d: 75.9% yield (yellowish syrup). R_(f) (hexanes/AcOEt 1:1) 0.10. ¹ HNMR (CDCl₃) δ1.96 (s, 3H, 5-CH₃), 2.34 (ddd, 1H, J=14.7, 7.9, 4.6 Hz,3'-H.sub.α), 2.37 (br s, 1H, OH, D₂ O exchangable), 2,62 (dd, 1H,J=14.7, 6.8 Hz, 3'-H.sub.β), 3.44-4.01 (m, 24H), 4.72 (s, 2H, OCH₂ Ph),5.25 (s, 1H, 5'-H), 5.48 (d, 1H, J=4.6 Hz, 4'-H), 5.52 (s, 2H, NCH₂ O),6.79 (dd, 1H, J=7.9, 6.8 Hz, 2'-H), 7.25-7.65 (m, 9H, 6-H andaromatic-H), 7.98-8.06 (m, 2H, aromatic-H). ¹³ C NMR (CDCl₃) δ13.03,34.85 (3'-C), 61.28, 67.83, 70.12, 70.33, 71.38, 71.96, 72.47, 77.44(4'-C), 86.22 (2'-C), 106.27 (5'-C), 111.08 (5-C), 127.39, 128.08,128.44 (6-C), 128.94, 129.60, 133.54, 134.38, 137.92, 151.17 (2-C),163.08 (4-C), 165.29 (benzoyl C═O).

EXAMPLE 9 (2'R,4'S,5'S)-(4'-Hydroxy-5'-O-polyethyleneglycoltetrahydrofuran-2'-yl)thymine (10)

A suspension of 4.5 mmol of 9 and 2-3 g of Pd(OH)₂ /C in 40 mL of 3:1MeOH/acetone was stirred at 23° C. under H₂ atmosphere for 16 h. Thesuspended material was filtrated through a pad of Celite in a Buchnerfunnel and the solid in the funnel was rinsed several times withacetone. The combined filtrate and rinses were concentrated at reducedpressure to give a light yellow syrup. The syrup was redissolved in 25mL of MeOH and 0.3 g (7.5 mmol) of NaOH was added. The mixture wasstirred at 23° C. for 6 h and concentated at reduced pressure. Theresidue was purifed by SiO₂ column chromatography to yield 10.

10a: yield 88.3% (white solid). mp 210° C. (dec.). R_(f) (CHCl₃ /MeOH9:1) 0.22. ¹ H NMR (D₂ O) δ1.84 (s, 3H, 5-CH₃), 2.34 (m, 2H, 3'-H),3.57-3.83 (m, 4H), 4.42 (dd, J=3.7, 2.1 Hz, 4'-H), 5.08 (s, 1H, 5'-H),6.56 (t, 1H, J=7.1 Hz, 2'-H), 7.56 (s, 1H, 6-H). ¹³ C NMR (DMSO-d₆)δ12.07, 36.94 (3'-C), 59.98, 69.33, 74.11 (4'-C), 84.99 (2'-C), 108.50(5'-C), 110.43 (5-C), 135.96 (6-C), 150.71 (2-C), 163.67 (4-C). AnalCalcd for C₁₁ H₁₆ N₂ O₆.0.7CH₃ OH: C, 47.69; H, 6.38; N, 9.51. Found: C,47.70; H, 6.01; N, 9.52.

10b: yield 90.6% (white solid). mp 54°-56° C. R_(f) (CHCl₃ /MeOH 9:1)0.41. ¹ H NMR (CDCl₃) δ1.93 (s, 3H, 5-CH₃), 2.11 (ddd, 1H, J=14.2, 7.9,4.9 Hz, 3'-H.sub.α), 2.33 (br s, 1H, 4'-OH), 2,41 (dd, 1H, J=14.2, 6.5Hz, 3'-H.sub.β), 3.38 (s, 3H, CH₃ O), 3.54-3.94 (m, 12H), 4.37 (d, 1H,J=4.9 Hz, 4'-H), 5.14 (s, 1H, 5'-H), 6.68 (dd, 1H, J=7.9, 6.5 Hz, 2'-H),7.45 (s, 1H, 6-H), 9.25 (s, 1H, NH). ¹³ C NMR (CDCl₃) δ12.50, 37.42(3'-C), 58.84 (CH₃ O), 67.52, 70.25, 70.40, 71.71, 75.01 (4'-C), 85.90(2'-C), 109.23 (5'-C), 111.60 (5-C), 136.29 (6-C), 151.10 (2-C), 164.40(4-C). Anal Calcd for C₁₆ H₂₆ N₂ O₈.2H₂ O: C, 46.83; H, 7.32; N, 6.83.Found: C, 46.94; H, 7.50; N, 6.74.

10c: yield 85.9% (white foam). R_(f) (CHCl₃ /MeOH 9:1) 0.36. ¹ H NMR(CDCl₃) δ1.92 (s, 3H, 5-CH₃), 2.09 (ddd, 1H, J=14.2, 7.8, 4.9 Hz,3'-H.sub.α), 2,41 (dd, 1H, J=14.2, 6.6 Hz, 3'-H.sub.β), 3.58-3.90 (m,16H), 4.36 (d, 1H, J=4.9 Hz, 4'-H), 5.20 (s, 1H, 5'-H), 6.63 (dd, 1H,J=7.8, 6.6 Hz, 2'-H), 7.46 (s, 1H, 6-H), 8.87 (s, 1H, NH). ¹³ C NMR(CDCl₃) δ12.53, 37.35 (3'-C), 61.26, 67.47, 70.00, 70.35, 72.51, 74.99,77.71 (4'-C), 85.90 (2'-C), 109.27 (5'-C), 111.47 (5-C), 136.33 (6-C),151.05 (2-C), 164.45 (4-C). Anal Calcd for C₁₇ H₂₈ N₂ O₉.0.5 H₂ O: C,49.39; H, 7.02; N, 6.78. Found: C, 49.58; H, 7.12; N, 6.66.

10d: yield 87.3% (white wax). R_(f) (CHCl₃ /MeOH 10:1) 0.40. ¹ H NMR(CDCl₃) δ1.92 (s, 3H, 5-CH₃), 2.08 (ddd, 1H, J=13.8, 7.7, 4.8 Hz,3'-H.sub.α), 2,39 (dd, 1H, J=13.8, 6.5 Hz, 3'-H.sub.β), 2.81 (s, 1H,4'-OH), 3.32-3.92 (m, 24H), 4.01 (s, 1H, OH), 4.37 (d, 1H, J=4.8 Hz,4'-H), 5.16 (s, 1H, 5'-H), 6.66 (dd, 1H, J=7.8, 6.5 Hz, 2'-H), 7.45 (s,1H, 6-H), 8.90 (s, 1H, NH). ¹³ C NMR (CDCl₃) δ12.49, 37.50 (3'-C),61.40, 67.53, 70.14, 70.41, 72.53, 75.01 (4'-C), 85.92 (2'-C), 109.27(5'-C), 111.50 (5-C), 136.20 (6-C), 151.00 (2-C), 164.25 (4-C). AnalCalcd for C₂₁ H₃₆ N₂ O₁₁.0.75 H₂ O: C, 49.85; H, 7.42; N, 5.54. Found:C, 49.79; H, 7.50; N, 5.60.

EXAMPLE 10 (2'R,4'S,5'S)-(4'-(2-Cyanoethyl-N,N-diisopropylaminophosphorityl)-5'-O-(dimethoxytrityloxytetraethyleneglycol)tetrahydrofuran-2'-yl)thymine (12)

A solution of 1.3 g (3.2 mmol) of 10c, 1.0×10⁻² g (8.2×10⁻² mmol) ofDMAP, and 1.0 g (10.0 mmol) of Et₃ N in 30 mL of pyridine was treatedwith 1.6 g (4.8 mmol) of DMTr-Cl at 23° C. for 14 h. The solvent wasremoved at reduced pressure and the residue was purified by SiO₂ columnchromatography (CHCl₃ /MeOH 20:1 then 10:1) gave 1.9 g (83.6%) of 11 asa yellow foam. R_(f) (CHCl₃ /MeOH 20:1) 0.23. ¹ H NMR (CDCl₃) δ1.92 (s,3H, 5-CH₃), 2.04 (ddd, 1H, J=14.2, 7.6, 4.8 Hz, 3'-H.sub.α), 2,32 (dd,1H, J=14.2, 7.0 Hz, 3'-H.sub.β), 3.23 (t, 2H, J=5.0 Hz), 3.29 (br s, 1H,4'-OH), 3.59-3.89 (m, 14H), 3.79 (s, 6H, 2×OCH₃), 4.31 (d, 1H, J=4.8 Hz,4'-H), 5.10 (s, 1H, 5'-H), 6.62 (dd, 1H, J=7.6, 7.0 Hz, 2'-H), 6.82 (d,4H, J=8.9 Hz), 7.20-7.48 (m, 10H, 6-H and aromatic-H), 8.38 (s, 1H, NH).¹³ C NMR (CDCl₃) δ12.69, 37.58 (3'-C), 55.26, 63.15, 67.74, 70.73, 75.24(4'-C), 86.01 (2'-C), 86.08, 109.49 (5'-C), 111.80 (5-C), 113.12,126.74, 127.68, 127.83, 128.24, 130.13, 136.31 (6-C), 145.13, 151.24(2-C), 158.41, 164.44 (4-C).

A solution of 1.8 g (2.6 mmol) of 11 and 3×10⁻² g (1.8×10⁻¹ mmol) ofN,N-diisopropylammonium tetrazolide in 50 mL of CH₂ Cl₂ was treated with1.1 g (3.6 mmol) of 2-cyanoethoxy-N,N-diisopropylphosphoramidite at 23°C. for 16 h. The mixture was then diluted with 100 mL CH₂ Cl₂ and washedwith sat. aqueous NaHCO₃ (30 mL) and brine (30 mL). The organic layerwas dried (MgSO₄) and concentrated at reduced pressure. The residue waspurified by SiO₂ column chromatography (0.5% Et₃ N in AcOEt) gave 1.9 g(77.9%) of 12 as a slightly yellow foam. ³¹ P NMR (CDCl₃) δ149.33 and149.53 ppm.

EXAMPLE 11 2-(2-Phthalimidoethoxy)ethanol (13)

A suspension of 30.0 g (202.5 mmol) of phthalic anhydride, 20 mL (21.0g, 199.4 mmol) of 2-(2-aminoethoxy)-ethanol in 400 mL of benzene wasadded 1.0×10⁻² g (5.3×10⁻⁵ mmol) of TsOH.H₂ O. The reaction flask wasconnected to a Dean-Stark condenser and then heated to reflux for 14 h.The solvent was removed at reduced pressure and the residue was purifiedby SiO₂ column chromatography (hexanes/AcOEt 1:1 then CHCl₃ /MeOH 20:1)gave 43.7 g (93.1%) of 13 as a light yellow syrup. This syrup slowlycrystallized on standing at room temperature overnight as needle-likecrystals. mp 66°-66.5° C. R_(f) (hexanes/AcOEt 1:1) 0.17. ¹ H NMR(CDCl₃) d 2.41 (t, 1H, J=6.2 Hz, OH), 3.57-3.93 (m, 8H), 7.69-7.87 (m,4H). ¹³ C NMR (CDCl₃) d 37.52, 61.62, 68.24, 72.24, 123.36, 131.97,134.00, 168.38 (C═O). Anal Calcd for C₁₂ H₁₃ NO₄ : C, 61.28; H, 5.53; N,5.96. Found: C, 61.34; H, 5.62; N, 5.94.

EXAMPLE 12(2'R,4'S,5'S)-4'-Benzoxy-5'-O-((2-(2-phthalimidoethoxy)ethyl)-tetrahydrofuran-2'-yl)-N³-(benzoxymethyl)thymine (14)

A solution of 10.8 g (22.0 mmol) of 8, 10.0 g (42.6 mmol) of 13, and 5.7g (8.0 mL, 57.0 mmol) of Et₃ N in 200 mL of CH₂ CL₂ was chilled to -23°C. To this solution, 16.9 g (14.6 mL, 76.0 mmol) of TMSOTf was added inone portion. The resultant mixture was stirred at -23° C. for 15 min andthen standed in the freezer (-15° C.) for 16 h. The reaction wasquenched by pouring into 1 L, 4:1 mixture of AcOEt/H₂ O containing 20 mLof Et₃ N. The organic layer was washed with H₂ O (300 mL), dried(MgSO₄), and concentrated at reduced pressure. The residue was purifiedby SiO₂ column chromatography (hexanes/AcOEt 1:1) gave 12.4 g (84.8%) of14 as very hydroscopic white foam. R_(f) (hexanes/AcOEt 1:1) 0.22. ¹ HNMR (CDCl₃) δ1.89 (s, 3H, 5-CH₃), 2.30 (ddd, 1H, J=13.2, 8.3, 4.6 Hz,3'-H.sub.α), 2.57 (dd, 1H, J=13.2, 6.5 Hz, 3'-H.sub.β), 3.63-4.01 (m,8H), 4.71 (s, 2H, OCH₂ Ph), 5.19 (s, 1H, 5'-H), 5.36 (d, 1H, J=4.6 Hz,4'-H), 5.48 (s, 2H, NCH₂ O), 6.72 (dd, 1H, J=8.3, 6.5 Hz, 2'-H),7.28-8.01 (m, 13H, 6-H and aromatic-H), 8.02 (dd, 2H, J=7.8, 1.5 Hz,aromatic-H). ¹³ C NMR (CDCl₃) δ13.01 (5-CH₃), 34.87 (3'-C), 36.91,67.64, 67.78, 69.34, 70.62, 72.05, 73.60 (4'-C), 86.30 (2'-C), 106.16(5'-C), 111.13 (5-C), 123.08, 127.51, 127.76, 128.21, 128.50, 129.20,129.70, 131.92, 133.54 (6-C), 134.00, 134.41, 138.08, 151.26 (2-C),163.07 (4-C), 165.29 (phthalimido C═O) 168.02 (benzoxy C═O). Anal Calcdfor C₃₆ H₃₅ N₃ O₁₀. 0.7 CHCl₃ : C, 58.51; H, 4.74; N, 5.58. Found: C,58.78; H, 4.74; N, 5.60.

EXAMPLE 13(2'R,4'S,5'S)-4'-Hydroxy-5'-O-((2-(2-aminoethoxy)ethyl)-tetrahydrofuran-2'-yl)thymine(15)

A suspension of 12.4 g (18.5 mmol) of 14 and 7.6 g of 10% Pd(OH)₂ /C in100 mL of 1:1 mixture of acetone/MeOH was stirred under atmosphere H₂for 16 h. The suspended material was filtered through a pad of Celiteand the filtrate was concentrated at reduced pressure to give 7.8 g(76.7%) of de-BOM product as a white form. The de-BOM product wasre-dissolved in 100 mL MeOH and 1.54 g (48.0 mmol) of H₂ NNH₂ was added.The mixture was heated to reflux for 16 h and then concentrated atreduced pressure. The resultant crude, free amine product (˜8 g) wasredissolved in 100 mL MeOH and 4.0 g (100.0 mmol) of NaOH was added. Themixture was stirred at 45° C. for 4 h and then the solvent was removedat reduced pressure. The residue was purified by SiO₂ columnchromatography (2% Et₃ N in CHCl₃ /MeOH 1:1) to give 2.2 g (37.7% from14) of 15 as a white solid. R_(f) (2% Et₃ N in CHCl₃ /MeOH 1:1) 0.21. ¹H NMR (CD₃ OD) δ1.93 (s, 3H, 5-CH₃), 2.27 (m, 2H, 3'-H), 2.82 (m, 2H),3.58-3.93 (m, 6H), 4.30 (d, 1H, J=4.5 Hz, 4'-H), 5.05 (s, 1H, 5'-H),6.62 (t, 1H, J=8.1 Hz, 2'-H), 7.58 (s, 1H, 6-H). ¹³ C NMR (CD₃ OD)δ12.80 (5-CH₃), 38.33 (3'-C), 51.23, 68.77, 70.00, 71.31, 76.18 (4'-C),87.29 (2'-C), 110.77 (5'-C), 112.37 (5-C), 137.84 (6-C), 152.75 (2-C),166.31 (4-C).

EXAMPLE 14 (2'R,4'S,5'S)-(4'-Hydroxy-5'-O-((2-aminoethoxy)ethyl)-tetrahydrofuran-2'-yl)thymine, cholesterylforamide(16)

A stirred solution of 2.1 g (6.8 mmol) of 15 in 20 mL of 1:1 CH₂ Cl₂/pyridine was added a solution of 3.3 g (7.5 mmol) of cholesterylchloroformate in 20 mL CH₂ Cl₂ at 0° C. over 45 min period. Theresultant mixture was stirred for additional 3 h at 0° C. and thendiluted with 200 mL CH₂ Cl₂, washed with sat. aqueous NaHCO₃ (50 mL) andbrine (50 mL). The organic layer was dried (MgSO₄) and concentrated atreduced pressure. The residue was purified by SiO₂ column chromatography(CH₂ Cl₂ /AcOEt 9:1 then AcOEt) to give 2.2 g (45.4%) of 16 as a whitesolid. R_(f) (AcOEt) 0.44. ¹ H NMR (DMSO-d₆, assignment for somecharacteristic proton resonances): δ4.93 (s, 1H, 5'-H), 5.33 (d, 1H,J=3.8 Hz, 4'-OH), 5.45 (d, 1H, J=3.8 Hz, 4'-H), 6.48 (t, 1H, J=8.2 Hz,2'-H), 8.37 (s, 1H, 6-H), 11.38 (s, 1H, NH). --C ^(NMR) (DMSO-d₆) d11.65, 12.18, 18.55, 18.98, 22.37, 22.61, 23.38, 23.89, 27.43, 27.83,31.42, 34.72, 34.87, 35.30, 35.78, 36.10, 41.89, (3'-C), 47.38, 49.56,55.74, 56.20, 66.82, 69.15, 73.01, 73.88, 74.11 (4'-C), 79.20, 85.09(2'-C), 108.61 (5'-C), 110.34 (5-C), 139.73 (6-C), 150.67 (2-C), 154.99(formamide C═O), 163.55 (4-C).

EXAMPLE 15 (2'R,4'S,5'S)-(4'-O-(2-Cyanoethoxy-N,N-diisopropylamino-phosphorityl)-5'-O-((2-aminoethoxy)ethyl)-tetrahydrofuran-2'-yl) thymine,cholesterylforamide (17)

A solution of 1.7 g (2.3 mmol) of 16 and 1.4 g (1.9 mL, 11.0 mmol) ofi-Pr₂ NEt in 40 mL CH₂ Cl₂ was treated with 1.1 g (1.0 mL, 4.6 mmol) of2-cyanoethyl N,N-diisopropylchlorophosphoramidite at room temperaturefor 24 h. The resultant mixture was diluted with 100 mL CH₂ Cl₂ andwashed with sat. aqueous NaHCO₃ (30 mL) and brine (30 mL). The organiclayer was dried (MgSO₄) and concentrated at reduced pressure. Theresidue was purified by SiO₂ column chromatography (0.5% Et₃ N inhexanes/AcOEt 1:1 then AcOEt) to give 0.36 g (16.6%) of 17 as a whitefoam. ³¹ P NMR (CDCl₃) δ149.23, 149.43 ppm.

A summary of the synthesis of compounds 8 through 17 are presented onthe following pages. ##STR12##

EXAMPLE 16 N³-Benzoxymethyl-(4'-O-benzoyl-5'-O-adamantyl-tetrahydrofuran-2'-yl)thymine(18)

To a solution of 8 (3.5 g, 7.1 mmol) and 1-adamantanol (2.2 g, 14.2mmol) in CH₂ Cl₂ (100 ml) was added TMSOTf (2.7 ml, 3.2 g, 14.2 mmol) at-23° C. in one portion via a syringe. The resultant mixture was stirredat -23° C. for 0.5 hr and then placed in a freezer (-15° C.) for 18 hr.The reaction mixture was then poured into a 40:10:1 bilayer solution ofAcOEt/H₂ O/Et₃ N (500 ml), the organic layer dried over MgSO₄ andconcentrated at reduced pressure. The residue was purified by SiO₂column chromatography (hexanes/AcOEt 4:1 then 2:1) to give 3.6 g (86.7%)of the title compound as a white solid. R_(f) (hexanes/AcOEt 1:1) 0.60.¹ H NMR (CDCl₃) δ1.50-1.95 (m, 12H, adamantyl-H), 1.98 (s, 3H, 5-CH₃),2.05-2.25 (M, 4H, adamantyl-H), 2.3 (ddd, 1H, J=14.5, 7.9, 4.5 Hz,3'-H.sub.α), 2.63 (DD, 1H, J=14.5, 6.5 Hz, 2'-H.sub.β), 4.73 (s, 2H,OCH₂ Ph), 5.27 (d, 1H, J=4.5 Hz, 4'-H), 5.52 (s, 2H, NCH₂ O), 5.61 (s,1H, 5'-H), 6.71 (dd, 1H, J=7.9, 6.5 Hz, 2'-H), 7.2-7.65 (m, 8H,aromatic-H), 7.78 (s, 1H, 6-H), 8.08 (d, 2H, J=7.6 Hz, aromatic-H). ¹³C-NMR (CDCl₃) δ13.37, 30.58, 35.42 (3'-C), 42.66, 45.33, 70.65, 72.20,75.96, 79.14 (4'-C) 86.31 (2'-C), 99.48 (5'-C), 110.54 (5-C), 127.61,128.28, 129.25, 129.83, 133.61, 135.36 (6-C), 138.14, 151.37 (2-C),163.36 (4-C), 165.77 (benzoyl C═O).

EXAMPLE 17 Preparation of Oligonucleotides

Oligonucleotides are prepared utilizing normal protocol for solid stateDNA synthesis, (see Oligonucleotide synthesis, a practice approach, Ed.M. J. Gait, IRL Press 1984, Oxford University Press, New York). DMTprotected phosphoramidite nucleotides are added to the growingoligonucleotide structure on a solid state support in the normal manner.Coupling efficiencies are typically greater than 96% for each step ofthe synthesis with the overall coupling efficiency greater than 91% forthe oligomer. The resulting oligomers are characterized by both gelchromatography and by HPLC using standard protocols.

EXAMPLE 18 Preparation of Oligonucleotides Containing 5'-TerminalNucleotides Having 4'-Desmethyl Structure Bearing A Conjugation Molecule

An oligonucleotide of the desired sequence is prepared as per thepreceding Example 17 in the normal 3' to 5' direction. The lastnucleotide of the sequence is preformed to include the desired4'-desmethyl structure bearing a conjugate group, e.g. compounds 12 and17, attached via linker groups and compound 18, directly attached,thereon as per the above Examples 10, 15 and 17. This last nucleotideunit, as a 3'-phosphoamidite, is coupled to the remainder of theoligonucleotide sequence utilizing normal protocol for solid state DNAsynthesis, as above. As with the remainder of the sequences, couplingefficiencies are generally greater than 96% for this step of thesynthesis.

A typical synthesis was that of an oligonucleotide of the sequence:

T*GC ATC CCC CAG GCC ACC AT, SEQ ID NO. 1, where T* is the nucleotide ofcompound 12, compound 17 or compound 18 above. The sequences wassynthesizer up to and including the penultimate nucleotide unit. Theconjugate group bearing, 4-desmethyl, terminal nucleotide, e.g. compound12, was then used as the ultimate nucleotide unit. The oligonucleotidewas removed from the solid support and purified by HPLC in the standardmanner.

EXAMPLE 18 Assay for Separating Expression of Intercellular AdhesionMolecule From Vascular Adhesion Molecules Using Selective ProteinInhibition

Various adhesion molecules are known that are associated with certainadverse events in biological systems. The study of these molecules canbe compounded by overlapping activities. Since the intercellularadhesion molecule, ICAM-1, and the vascular adhesion molecule, VCAM-1,both are capable of expressing certain similar properties, e.g. celladhesion or clumping, segregation of certain of their properties, onefrom the other, would assist in the evaluation of the properties of asingular adhesion molecule. This cellular assay isolates cytokineinduced protein induction of ICAM-1 from that of VCAM-1. The assaydifferentiates specific protein inhibition of ICAM-1 expression fromVCAM-1 expression via use of a probe oligonucleotide having a sequencecomplementary to the ICAM-1 messenger RNA. Inhibition of the inductionof ICAM-1 messenger RNA is effected by treatment of the cells with theprobe oligonucleotide in presence of a cationic liposome formulation(DOTMA/DOPE). Cells showing positive adhesion molecule expression inresponse to cytokine treatment are identified by increases in basallevels of the adhesion molecule protein after treatment with acombination of human TNF-α and murine INF-τ using a fluorescenceactivated cell sorter to identify the cells stained with fluorescentantibodies to either the ICAM-1 or VCAM-1 expressed proteins.

The oligonucleotide utilized as the probe was an oligonucleotide of theinvention of the sequence T*GC ATC CCC CAG GCC ACC AT, SEQ ID NO. 1,complementary to the 3' untranslated region of murine ICAM-1 messengerRNA. It includes a conjugation PEG molecule (compound 12 above) as the5' terminal T nucleotide (nucleotide T* in the above sequence). Thisoligonucleotide is then used to differentiate ICAM-1 expression fromVCAM-1 expression in the test protocol. In cell lines from otherspecies, the base sequence of conjugated oligonucleotide of theinvention used in the test is synthesized to be of a sequencecomplementary to a portion of the ICAM-1 messenger RNA for the speciesof interest.

A murine endothelioma cell line designated bEND.3, supplied by Dr.Werner Risau, Max-Planck-Institute, Planegg-Martinsreid, Germany, istested for adhesion molecule expression as follows. The cells weretreated with nanomolar concentrations of the conjugated oligonucleotide,SEQ ID NO. 1, in the presence of 15 μg/ml DOTMA/DOPE liposome(Lipofectin) for 4 hours in a serum free media (Opti-MEM serum-freemedium, GIBCO, Grand Island, N.Y.) in the manner described by Chiang etal., J. Biol. Chem., 1991, 266, 18162-18171 or Bennett, et al., J.Immunol, 1994, 152, 3530-3540. The media was aspirate and adhesionmolecule expression was induced with human TNF-α (5 ng/ml, R&D Systems,Minneapolis, Minn.) and murine IFN-τ (1000 μ/ml, Genzyme, Cambridge,Mass.) overnight in DMEM high glucose with 10% fetal bovine serum(Hyclone, Logan, Utah). The cells were trypsinized and washed. The cellswere stained with ICAM-1-PE fluorescent antibodies (anti-ICAM-1mAB,PharMingen, San Diego, Calif.) and VCAM-1-FITC fluorescent antibodies(anti-VCAM-1 mAB, PharMingen, San Diego, Calif.) and read on afluorescence activated cell sorter.

Concurrent treatment of the same bEND.3 cell line absent the conjugatedoligonucleotide served as a positive control. Both ICAM-1 and VCAM-1induction were observed in the positive control and normalized to 100%.The conjugated oligonucleotide was used at 0.1, 0.3 and 0.5 uM and thelevels of both of induced expression ICAM-1 and VCAM-1 were compared tothis positive control. Inhibition of VCAM-1 expression was observed tobe 90.08%, 103.82% and 96.33% of control for the 0.1, 0.3 and 0.5 uMconcentrations, respectively whereas inhibition of ICAM-1 expression was3.77%, 4.40% and 8.74% of control for the 0.1, 0.3 and 0.5 uMconcentrations, respectively.

As these results show, in the murine bEND.3 cell line both ICAM-1 andVCAM-1 expression were induced in the positive control. However, in thepresence of the ICAM-1 specific messenger RNA complementaryoligonucleotide of the invention, VCAM-1 protein expression wasmaintained but ICAM-1 protein expression was inhibited. Thus the VCAM-1mAB,

56 PATENT expression of VCAM-1 is disconnect from that of ICAM-1 suchthat VCAM-1 expression can be examined independent of ICAM-1 expression.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 1                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       TGCATCCCCCAGGCCACCAT20                                                        __________________________________________________________________________

What is claimed is:
 1. A compound having the structure: ##STR13##wherein: R_(L) is a group of formula:

    R.sub.C --[Y].sub.e --Z--

wherein: Z is O, S or HN; Y is a bivalent linker; e is 0 or 1; R_(C) isalkyl, alkenyl, alkynyl, a polyamine, a polyether, a polythioether, asteroid molecule, a reporter molecule, an aromatic lipophilic molecule,a non-aromatic lipophilic molecule, a reporter enzyme, a peptide, aprotein, a water soluble vitamin, a lipid soluble vitamin, acarbohydrate, a terpene molecule, a phospholipid, an intercalator, acell receptor binding molecule, a crosslinking agent, or a porphyrin;B_(x) is a nucleobase; X is H, OH, O-alkyl, O-alkoxyalkyl, O-alkylamino,or F; Q is O, S, CH₂, CHF or CF₂ ; and R_(D) is H, hydroxyl, anactivated phosphorous group, a nucleoside, an activated nucleotide, anucleotide, an oligonucleotide, an oligonucleoside or a protectedderivative thereof.
 2. The compound having the structure: ##STR14##wherein: L is a group of formula:R₇ --[W]_(a) --[S--(CH₂)_(q) ]_(u)--[HN--(CH₂)_(n) ]_(t) --.sub.[O--(CH₂)_(m) ]_(v) --Z--wherein: t, u andv are each independently integers from 0 to 200; m, n and q are eachindependently integers from 1 to 4; Z is S, O or HN; R₇ is R_(c), H, ora protecting group; B_(x) is a nucleobase; X is H, OH, O-alkyl,O-alkoxylalkyl, O-alkylamino, or F; Q is O; W is the residue of alinking moiety, said linking moiety being selected from the groupconsisting of acid chlorides, anhydrides, cyclic anhydrides, alkylhalides, organometallics, chloroformates, isocynates, hydrazines, acids,hydroxylamines, semicarbazides, thiosemicarbazides, hydrazones,hydrazides, trityl thiol, oximes, hydrazide-hydrazones, semicarbazonesand semithiocarbazones; a is 0 or 1; R_(D) is H, hydroxyl, an activatedphosphorous group, a nucleoside, an activated nucleotide, a nucleotide,an oligonucleotide, an oligonucleoside or a protected derivativethereof; R_(C) is alkyl, alkenyl, alkynyl, a polyamine, a polyether, apolythioether, a steroid molecule, a reporter molecule, an aromaticlipophilic molecule, a non-aromatic lipophilic molecule, a reporterenzyme, a peptide, a protein, a water soluble vitamin, a lipid solublevitamin, a carbohydrate, a terpene molecule, a phospholipid, anintercalator, a cell receptor binding molecule, a crosslinking agent, ora porphyrin; and provided that t, u and v are not all simultaneously 0.3. The compound of claim 2 wherein t and u are
 0. 4. The compound ofclaim 2 wherein t and u are 0, and m is
 2. 5. The compound of claim 2wherein u and v are
 0. 6. The compound of claim 2 wherein u and v are 0and n is
 2. 7. The compound of claim 2 wherein X is H or OH.
 8. Thecompound of claim 2 wherein R_(D) is H or OH.
 9. The compound of claim 2wherein R_(D) is an activated phosphorous group.
 10. A compound of claim2 wherein R_(D) is an oligonucleotide.
 11. The compound of claim 2wherein t and v are
 0. 12. The compound of claim 2 wherein t and v are 0and q is
 2. 13. The compound of claim 2 wherein Z is O or N.
 14. Thecompound of claim 2 wherein R_(C) is asteroid molecule.
 15. A compoundof claim 14 wherein the steroid molecule is cholic acid, deoxycholicacid, dehydrocholic acid, cortisone, digoxigenin, testosterone,cholesterol or 3-trimethylaminomethylhydrazido cortisone.
 16. Thecompound of claim 2 wherein R_(C) is a water soluble vitamin.
 17. Thecompound of claim 16 wherein the water soluble vitamin is thiamine,riboflavin, nicotinic acid, pyridoxal phosphate, pyridoxine,pyridoxamine, deoxypyridoxine, pantothenic acid, biotin, folic acid,5'-deoxyadenosylcobalamin, inositol, choline or ascorbic acid.
 18. Thecompound of claim 2 wherein R_(C) is a lipid soluble vitamin.
 19. Thecompound of claim 18 wherein the lipid soluble vitamin is a retinal, aretinol, retinoic acid, β-carotene, vitamin D, cholecalciferol, atocopherol, or a phytol.
 20. The compound of claim 2 wherein R_(C) is aprotein.
 21. The compound of claim 20 wherein the protein is aphosphodiesterase, a peroxidase, a phosphatase or a nuclease.
 22. Thecompound of claim 2 wherein R_(C) is a reporter molecule.
 23. Thecompound of claim 22 wherein the reporter molecule is a chromaphore, afluorophore or a radiolabel-containing moiety.
 24. The compound of claim23 wherein the fluorophore is fluorescein, chrysine, anthracene,perylene, pyrene, rhodamine.
 25. The compound of claim 2 wherein X is F,O-alkyl having from one to six carbons, O-alkoxyalkyl having from 2 to 6carbons, or O-alkylamino having from one to six carbons.
 26. Thecompound of claim 2 wherein R_(D) is H or OH.
 27. The compound havingthe structure: ##STR15## wherein: R_(C) is O-alkyl, O-alkenyl,O-alkynyl, a polyamine, a polyether, a steroid molecule, a reportermolecule, an aromatic lipophilic molecule, a non-aromatic lipophilicmolecule, a reporter enzyme, a peptide, a protein, a water solublevitamin, a lipid soluble vitamin, a carbohydrate, a terpene molecule, aphospholipid, an intercalator, a cell receptor binding molecule, acrosslinking agent, or a porphyrin;B_(x) is a nucleobase; X is H, OH,O-alkyl, O-alkoxyalkyl, O-alkylamino, or F; Q is O, S, CH₂, CHF or CF₂ ;and R_(D) is H, hydroxyl, an activated phosphorous group, a nucleoside,an activated nucleotide, a nucleotide, an oligonucleotide, anoligonucleoside or a protected protected derivative thereof.
 28. Thecompound of claim 27 wherein R_(C) is a polyether, a polyamine or anon-aromatic lipophilic molecule.
 29. The compound having the structure:##STR16## wherein: J is a leaving group;B_(x) is a nucleobase; X is H,OH, O-alkyl, O-alkoxylalkyl, O-alkylamino, or F; Q is O, S, CH₂, CHF orCF₂ ; and R_(D) is H, hydroxyl, an activated phosphorous group, anucleoside, an activated nucleotide, a nucleotide, an oligonucleotide,an oligonucleoside or a protected derivative thereof.
 30. The compoundof claim 29 wherein J is OH, SH, NH₂, trifluoromethylsulfonyl,methylsulfonyl, halogen, O-trichloroacetimidate, acyloxy, dialkylphosphite, 2,4,6-trichlorophenyl, p-toluenesulfonyl,4-dimethylaminoazobenzenesulfonyl or 5-dimethylaminonaphthalenesulfonyl.